U.S. patent number 9,655,892 [Application Number 14/938,647] was granted by the patent office on 2017-05-23 for certain chemical entities, compositions and methods.
This patent grant is currently assigned to Intellikine LLC. The grantee listed for this patent is Intellikine LLC. Invention is credited to Katrina Chan, Liansheng Li, Yi Liu, Pingda Ren, Christian Rommel, Troy Edward Wilson.
United States Patent |
9,655,892 |
Ren , et al. |
May 23, 2017 |
**Please see images for:
( Certificate of Correction ) ** |
Certain chemical entities, compositions and methods
Abstract
Chemical entities that modulate PI3 kinase activity,
pharmaceutical compositions containing the chemical entities, and
methods of using these chemical entities for treating diseases and
conditions associated with P13 kinase activity are described
herein.
Inventors: |
Ren; Pingda (San Diego, CA),
Liu; Yi (San Diego, CA), Wilson; Troy Edward (San
Marino, CA), Li; Liansheng (San Diego, CA), Chan;
Katrina (Fremont, CA), Rommel; Christian (La Jolla,
CA) |
Applicant: |
Name |
City |
State |
Country |
Type |
Intellikine LLC |
La Jolla |
CA |
US |
|
|
Assignee: |
Intellikine LLC (La Jolla,
CA)
|
Family
ID: |
43450336 |
Appl.
No.: |
14/938,647 |
Filed: |
November 11, 2015 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20160158239 A1 |
Jun 9, 2016 |
|
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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14296953 |
Jun 5, 2014 |
9216982 |
|
|
|
13403394 |
Jul 22, 2014 |
8785456 |
|
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|
12503776 |
Jun 5, 2012 |
8193182 |
|
|
|
PCT/US2009/000042 |
Jan 5, 2009 |
|
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PCT/US2009/000038 |
Jan 5, 2009 |
|
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|
61201146 |
Dec 5, 2008 |
|
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|
61194294 |
Sep 26, 2008 |
|
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|
61009971 |
Jan 4, 2008 |
|
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61201146 |
Dec 5, 2008 |
|
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61194294 |
Sep 26, 2008 |
|
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61009971 |
Jan 4, 2008 |
|
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|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P
25/00 (20180101); A61P 13/00 (20180101); A61P
11/00 (20180101); A61P 3/00 (20180101); A61K
31/196 (20130101); A61K 45/06 (20130101); C07D
473/34 (20130101); A61K 31/131 (20130101); A61N
5/10 (20130101); A61P 17/00 (20180101); A61P
19/00 (20180101); A61K 31/506 (20130101); A61P
19/08 (20180101); A61P 19/02 (20180101); A61P
35/00 (20180101); A61P 43/00 (20180101); A61K
39/39558 (20130101); A61P 37/00 (20180101); A61P
9/00 (20180101); C07D 487/04 (20130101); A61P
1/00 (20180101); A61K 31/4725 (20130101); A61K
31/52 (20130101); A61K 39/3955 (20130101); A61P
37/02 (20180101); A61K 31/5377 (20130101); A61P
5/00 (20180101); A61P 11/06 (20180101); A61P
27/00 (20180101); A61P 3/10 (20180101); A61P
17/06 (20180101); A61P 29/00 (20180101); A61P
35/02 (20180101); A61P 7/02 (20180101); A61P
37/06 (20180101); A61P 37/08 (20180101); A61P
7/00 (20180101); A61N 5/1007 (20130101); Y02A
50/30 (20180101); Y02P 20/55 (20151101); A61N
2005/109 (20130101) |
Current International
Class: |
A61K
31/52 (20060101); A61N 5/10 (20060101); A61K
39/395 (20060101); A61K 31/196 (20060101); A61K
31/131 (20060101); C07D 473/34 (20060101); A61K
45/06 (20060101); A61K 31/5377 (20060101); C07D
487/04 (20060101); A61K 31/506 (20060101); A61K
31/4725 (20060101) |
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|
Primary Examiner: Shameem; Golam M M
Assistant Examiner: Daniel; Laura
Attorney, Agent or Firm: Jones Day
Parent Case Text
This application is a continuation application of U.S. patent
application Ser. No. 14/296,953, filed on Jun. 5, 2014, which is a
continuation application of U.S. patent application Ser. No.
13/403,394, filed on Feb. 23, 2012, now U.S. Pat. No. 8,785,456,
which is a divisional application of U.S. patent application Ser.
No. 12/503,776, filed on Jul. 15, 2009, now U.S. Pat. No.
8,193,182, which is a continuation in part of International
Application No. PCT/US09/00038, filed on Jan. 5, 2009 and
International Application No. PCT/US09/00042, filed on Jan. 5,
2009, each of which claims the benefit of U.S. Provisional
Application Nos. 61/009,971 filed on Jan. 4, 2008, 61/194,294 filed
on Sep. 26, 2008, and 61/201,146 filed on Dec. 5, 2008. All of the
above-referenced patent applications are hereby incorporated by
reference in their entirety for all purposes.
The present application is being filed with a Sequence Listing
submitted as filename 12928-143-999_SeqListing.txt, of size 1,026
bytes, which was created on Apr. 20, 2015. The Sequence Listing is
incorporated herein by reference in its entirety.
Claims
What is claimed is:
1. A method of treating non-Hodgkin lymphoma for therapeutic
benefit in a subject, comprising administering to the subject an
effective amount of a compound of formula: ##STR00665## or a
pharmaceutically acceptable salt thereof, and further comprising
administering one or more second therapeutic agents or therapy
selected from chemotherapeutic agents, cytotoxic agents, and
radiation therapy.
2. The method of claim 1, wherein the second therapeutic agent or
therapy is an anti-CD20 antibody.
3. The method of claim 1, wherein the second therapeutic agent or
therapy is rituximab.
4. The method of claim 1, wherein the second therapeutic agent or
therapy is a nitrogen mustard.
5. The method of claim 1, wherein the second therapeutic agent or
therapy is chlorambucil.
6. The method of claim 1, wherein the second therapeutic agent or
therapy is chlorambucil, chlornaphazine, estramustine, ifosfamide,
mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,
novembichin, phenesterine, prednimustine, trofosfamide, uracil
mustard, fludarabine, or cyclophosphamide.
7. The method of claim 1, wherein the second therapeutic agent or
therapy is fludarabine, cyclophosphamide, or rituximab, or a
combination thereof.
8. A method of treating chronic lymphocytic leukemia for
therapeutic benefit in a subject, comprising administering to the
subject an effective amount of a compound of formula: ##STR00666##
or a pharmaceutically acceptable salt thereof, and further
comprising administering one or more second therapeutic agents or
therapy selected from chemotherapeutic agents, cytotoxic agents,
and radiation therapy.
9. The method of claim 8, wherein the second therapeutic agent or
therapy is an anti-CD20 antibody.
10. The method of claim 8, wherein the second therapeutic agent or
therapy is rituximab.
11. The method of claim 8, wherein the second therapeutic agent or
therapy is a nitrogen mustard.
12. The method of claim 8, wherein the second therapeutic agent or
therapy is chlorambucil.
13. The method of claim 8, wherein the second therapeutic agent or
therapy is chlorambucil, chlornaphazine, estramustine, ifosfamide,
mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,
novembichin, phenesterine, prednimustine, trofosfamide, uracil
mustard, fludarabine, or cyclophosphamide.
14. The method of claim 8, wherein the second therapeutic agent or
therapy is fludarabine, cyclophosphamide, or rituximab, or a
combination thereof.
15. A method of treating leukemia or lymphoma for therapeutic
benefit in a subject, comprising administering to the subject an
effective amount of a compound of formula: ##STR00667## or a
pharmaceutically acceptable salt thereof, and further comprising
administering one or more nitrogen mustards.
16. The method of claim 15, wherein the nitrogen mustard is
chlorambucil, chlornaphazine, cyclophosphamide, estramustine,
ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride,
melphalan, novembichin, phenesterine, prednimustine, trofosfamide,
or uracil mustard.
17. The method of claim 1, wherein the compound is ##STR00668##
18. The method of claim 8, wherein the compound is ##STR00669##
19. The method of claim 15, wherein the compound is
##STR00670##
20. The method of claim 1, wherein the second therapeutic agent or
therapy is an mTOR inhibitor.
21. The method of claim 8, wherein the second therapeutic agent or
therapy is an mTOR inhibitor.
Description
BACKGROUND OF THE INVENTION
The activity of cells can be regulated by external signals that
stimulate or inhibit intracellular events. The process by which
stimulatory or inhibitory signals are transmitted into and within a
cell to elicit an intracellular response is referred to as signal
transduction. Over the past decades, cascades of signal
transduction events have been elucidated and found to play a
central role in a variety of biological responses. Defects in
various components of signal transduction pathways have been found
to account for a vast number of diseases, including numerous forms
of cancer, inflammatory disorders, metabolic disorders, vascular
and neuronal diseases (Gaestel et al. Current Medicinal Chemistry
(2007) 14:2214-2234).
Kinases represent a class of important signaling molecules. Kinases
can generally be classified into protein kinases and lipid kinases,
and certain kinases exhibit dual specificities. Protein kinases are
enzymes that phosphorylate other proteins and/or themselves (i.e.,
autophosphorylation). Protein kinases can be generally classified
into three major groups based upon their substrate utilization:
tyrosine kinases which predominantly phosphorylate substrates on
tyrosine residues (e.g., erb2, PDGF receptor, EGF receptor, VEGF
receptor, src, abl), serine/threonine kinases which predominantly
phosphorylate substrates on serine and/or threonine residues (e.g.,
mTorC1, mTorC2, ATM, ATR, DNA-PK, Akt), and dual-specificity
kinases which phosphorylate substrates on tyrosine, serine and/or
threonine residues.
Lipid kinases are enzymes that catalyze the phosphorylation of
lipids. These enzymes, and the resulting phosphorylated lipids and
lipid-derived biologically active organic molecules, play a role in
many different physiological processes, including cell
proliferation, migration, adhesion, and differentiation. Certain
lipid kinases are membrane associated and they catalyze the
phosphorylation of lipids contained in or associated with cell
membranes. Examples of such enzymes include phosphoinositide(s)
kinases (such as PI3-kinases, PI4-Kinases), diacylglycerol kinases,
and sphingosine kinases.
The phosphoinositide 3-kinases (PI3Ks) signaling pathway is one of
the most highly mutated systems in human cancers. PI3K signaling is
also a key factor in many other diseases in humans. PI3K signaling
is involved in many disease states including allergic contact
dermatitis, rheumatoid arthritis, osteoarthritis, inflammatory
bowel diseases, chronic obstructive pulmonary disorder, psoriasis,
multiple sclerosis, asthma, disorders related to diabetic
complications, and inflammatory complications of the cardiovascular
system such as acute coronary syndrome.
PI3Ks are members of a unique and conserved family of intracellular
lipid kinases that phosphorylate the 3'-OH group on
phosphatidylinositols or phosphoinositides. The PI3K family
comprises 15 kinases with distinct substrate specificities,
expression patterns, and modes of regulation (Katso et al., 2001).
The class I PI3Ks (p110.alpha., p110.beta., p110.delta., and
p110.gamma.) are typically activated by tyrosine kinases or
G-protein coupled receptors to generate PIP3, which engages
downstream effectors such as those in the Akt/PDK1 pathway, mTOR,
the Tec family kinases, and the Rho family GTPases. The class II
and III PI3-Ks play a key role in intracellular trafficking through
the synthesis of PI(3)P and PI(3,4)P2. The PIKKs are protein
kinases that control cell growth (mTORC1) or monitor genomic
integrity (ATM, ATR, DNA-PK, and hSmg-1).
The delta (.delta.) isoform of class I PI3K has been implicated, in
particular, in a number of diseases and biological processes. PI3K
.delta. is expressed primarily in hematopoietic cells including
leukocytes such as T-cells, dendritic cells, neutrophils, mast
cells, B-cells, and macrophages. PI3K .delta. is integrally
involved in mammalian immune system functions such as T-cell
function, B-cell activation, mast cell activation, dendritic cell
function, and neutrophil activity. Due to its integral role in
immune system function, PI3K .delta. is also involved in a number
of diseases related to undesirable immune response such as allergic
reactions, inflammatory diseases, inflammation mediated
angiogenesis, rheumatoid arthritis, auto-immune diseases such as
lupus, asthma, emphysema and other respiratory diseases. Other
class I PI3K involved in immune system function includes PI3K
.gamma., which plays a role in leukocyte signaling and has been
implicated in inflammation, rheumatoid arthritis, and autoimmune
diseases such as lupus.
Downstream mediators of the PI3K signal transduction pathway
include Akt and mammalian target of rapamycin (mTOR). Akt possesses
a plckstrin homology (PH) domain that binds PIP3, leading to Akt
kinase activation. Akt phosphorylates many substrates and is a
central downstream effector of PI3K for diverse cellular responses.
One important function of Akt is to augment the activity of mTOR,
through phosphorylation of TSC2 and other mechanisms. mTOR is a
serine-threonine kinase related to the lipid kinases of the PI3K
family. mTOR has been implicated in a wide range of biological
processes including cell growth, cell proliferation, cell motility
and survival. Disregulation of the mTOR pathway has been reported
in various types of cancer. mTOR is a multifunctional kinase that
integrates growth factor and nutrient signals to regulate protein
translation, nutrient uptake, autophagy, and mitochondrial
function.
As such, kinases, particularly PI3Ks are prime targets for drug
development. There remains a need for PI3K inhibitors suitable for
drug development. The present invention addresses this need and
provides related advantages as well by providing new classes of
kinase inhibitors.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides compounds of Formula
I below or pharmaceutically acceptable salts thereof, wherein
##STR00001##
W.sub.d is heterocycloalkyl, aryl or heteroaryl;
B is alkyl or a moiety of Formula II;
##STR00002##
wherein W.sub.c is aryl, heteroaryl, heterocycloalkyl, or
cycloalkyl, and q is an integer of 0, 1, 2, 3, or 4;
X is absent or --(CH(R.sup.9)).sub.z--, and z is an integer of
1;
Y is absent, or --N(R.sup.9)--;
R.sup.1 is hydrogen, alkyl, alkenyl, alkynyl, alkoxy, amido,
alkoxycarbonyl, sulfonamido, halo, cyano, or nitro;
R.sup.2 is alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,
aryl, heteroaryl, heteroarylalkyl, alkoxy, amino, halo, cyano,
hydroxy or nitro;
R.sup.3 is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,
heterocycloalkyl, alkoxy, amido, amino, alkoxycarbonyl sulfonamido,
halo, cyano, hydroxy or nitro;
R.sup.5, R.sup.6, R.sup.7, and R.sup.8 are independently hydrogen,
alkyl, alkenyl, alkynyl, cycloalkyl, hetercycloalkyl, alkoxy,
amido, amino, acyl, acyloxy, sulfonamido, halo, cyano, hydroxy or
nitro; and
each instance of R.sup.9 is independently hydrogen, alkyl,
cycloalkyl, or heterocycloalkyl.
In some embodiments of the compounds of Formula I, when both X and
Y are present then Y is --NH--.
In some embodiments of the compounds of Formula I, X is absent or
is --(CH(R.sup.9)).sub.z--, and z is independently an integer of 1,
2, 3, or 4; and Y is absent, --O--, --S--, --S(.dbd.O)--,
--S(.dbd.O).sub.2--, --N(R.sup.9)--,
--C(.dbd.O)--(CHR.sup.9).sub.z--, --C(.dbd.O)--,
--N(R.sup.9)(C.dbd.O)--, --N(R.sup.9)(C.dbd.O)NH--, or
--N(R.sup.9)C(R.sup.9).sub.2--.
In some of the embodiments, X is --CH.sub.2--,
--CH(CH.sub.2CH.sub.3), or --CH(CH.sub.3)--.
In some embodiments, X--Y is --CH.sub.2--N(CH.sub.3),
--CH.sub.2--N(CH.sub.2CH.sub.3), --CH(CH.sub.2CH.sub.3)--NH-- or
--CH(CH.sub.3)--NH--.
In some embodiments, W.sub.d is a pyrazolopyrimidine of Formula
III(a), or purine of Formula III(b), Formula III(c) or Formula
III(d) below:
##STR00003##
wherein R.sup.a' if Formula III(d) is hydrogen, halo, phosphate,
urea, a carbonate, amino, alkyl, alkenyl, alkynyl, cycloalkyl,
heteroalkyl, or heterocycloalkyl; R.sup.11 of Formula III(a) is H,
alkyl, halo, amino, amido, hydroxy, or alkoxy, and R.sup.12 of
Formula III(a), Formula III(c) or Formula III(d) is H, alkyl,
alkynyl, alkenyl, halo, aryl, heteroaryl, heterocycloalkyl, or
cycloalkyl. In some embodiments, W.sub.d is a pyrazolopyrimidine of
Formula III(a), wherein R.sup.11 is H, alkyl, halo, amino, amido,
hydroxy, or alkoxy, and R.sup.12 is cyano, amino, carboxylic acid,
or amido.
In some embodiments, the compound of Formula I has the structure of
Formula IV:
##STR00004##
wherein R.sup.11 is H, alkyl, halo, amino, amido, hydroxy, or
alkoxy, and R.sup.12 is H, alkyl, alkynyl, alkenyl, halo, aryl,
heteroaryl, heterocycloalkyl, or cycloalkyl. In some embodiments,
the compound of Formula I has the structure of Formula W wherein
R.sup.11 is H, alkyl, halo, amino, amido, hydroxy, or alkoxy, and
R.sup.12 is cyano, amino, carboxylic acid, or amido.
In some embodiments of the compound of Formula IV, R.sup.11 is
amino. In some embodiments of the compound of Formula IV, R.sup.12
is alkyl, alkenyl, alkynyl, heteroaryl, aryl, or heterocycloalkyl.
In some embodiments of the compound of Formula IV, R.sup.12 is
cyano, amino, carboxylic acid, amido, monocyclic heteroaryl, or
bicyclic heteroaryl.
In some embodiments of the compound of Formula I, the compound has
the structure of Formula V:
##STR00005##
In some of the embodiments of Formula V, NR.sup.9 is
--N(CH.sub.2CH.sub.3)CH.sub.2-- or N(CH.sub.3)CH.sub.2--.
In some of the embodiments of Formula I, the compound has a
structure of Formula VI:
##STR00006##
In some of the embodiments of the compound of Formula VI, R.sup.3
is --H, --CH.sub.3, --Cl, or --F, and R.sup.5, R.sup.6, R.sup.7,
and R.sup.8 are independently hydrogen.
In some of the embodiments of Formula VI, B is a moiety of Formula
II;
##STR00007## wherein W.sub.c is aryl, heteroaryl, heterocycloalkyl,
or cycloalkyl, and q is an integer of 0, 1, 2, 3, or 4.
In another aspect of the invention a compound and its
pharmaceutically acceptable salts having the structure of Formula
I-1 is provided, wherein:
##STR00008##
B is a moiety of Formula II;
wherein W.sub.c in B is aryl, heteroaryl, heterocycloalkyl, or
cycloalkyl, and q is an integer of 0, 1, 2, 3, or 4;
X is absent or --(CH(R.sup.9)).sub.z--, and z is an integer of
1;
Y is absent, or --N(R.sup.9)--;
when Y is absent, Wd is:
##STR00009## or when Y is present, Wd is:
##STR00010##
R.sup.1 is hydrogen, alkyl, alkenyl, alkynyl, alkoxy, amido,
alkoxycarbonyl, sulfonamido, halo, cyano, or nitro;
R.sup.2 is alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,
aryl, heteroaryl, heteroarylalkyl, alkoxy, amino, halo, cyano,
hydroxy or nitro;
R.sup.3 is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,
heterocycloalkyl, alkoxy, amido, amino, alkoxycarbonyl sulfonamido,
halo, cyano, hydroxy or nitro;
each instance of R.sup.9 is independently hydrogen,
C.sub.1-C.sub.10alkyl, cycloalkyl, or hetercyclooalkyl; and
R.sup.12 is H, alkyl, alkynyl, alkenyl, halo, aryl, heteroaryl,
heterocycloalkyl, or cycloalkyl.
In some embodiments, a compound of Formula I or Formula I-1 has the
structure of Formula IV-A:
##STR00011## In some embodiments of the compound of Formula IV-A,
R.sup.12 is substituted benzoxazole.
In some embodiments, a compound of Formula I or Formula I-1 has the
structure of Formula V-A:
##STR00012##
In some embodiments, a compound of Formula I or Formula I-1 has the
structure of Formula IV-A or Formula V-A.
In some embodiments, a compound of Formula I or Formula I-1 has the
structure of Formula V-B:
##STR00013##
In some embodiments, a compound of Formula I or Formula I-1 has the
structure of Formula VI-A:
##STR00014##
In some embodiments, a compound of Formula I or Formula I-1 is the
compound wherein B is a moiety of Formula II;
##STR00015## wherein W.sub.c is aryl, heteroaryl, heterocycloalkyl,
or cycloalkyl; q is an integer of 0 or 1; R.sup.1 is hydrogen,
alkyl, or halo; R.sup.2 is alkyl or halo; and R.sup.3 is hydrogen,
alkyl, or halo. In some embodiments, when both X and Y are present
then Y is --NH--. In other embodiments, R.sup.3 is --H, --CH.sub.3,
--CH.sub.2CH.sub.3, --CF.sub.3, --Cl or --F. In further
embodiments, R.sup.3 is methyl or chloro.
In some embodiments of the compound of Formula I or Formula I-1, X
is --(CH(R.sup.9)).sub.z--, wherein R.sup.9 is methyl and z=1; and
Wd is
##STR00016##
In other embodiments of the compound of Formula I or Formula I-1,
the compound is predominately in an (S)-stereochemical
configuration.
In further embodiments of the compound of Formula I or Formula I-1,
the compound has a structure of Formula V-A2:
##STR00017##
In some other embodiments of the compound of Formula I or Formula
I-1, R.sup.12 is a monocyclic heteroaryl, bicyclic heteroaryl, or
heterocycloalkyl.
In some other embodiments of the compound of Formula I or Formula
I-1, B is a moiety of Formula II:
##STR00018##
wherein W.sub.c is aryl or cycloalkyl.
In another aspect of the invention, a pharmaceutical composition is
provided which comprises a pharmaceutically acceptable excipient
and one or more compounds of any formulae provided herein,
including but not limited to Formula I, I-1, IV, IV-A, V, V-A,
V-A2, V-B, VI, and VI-A. In some embodiments, the composition is a
liquid, solid, semi-solid, gel, or an aerosol form.
In another aspect of the invention, a method of inhibiting a
phosphatidyl inositol-3 kinase (PI3 kinase), is provided
comprising: contacting the PI3 kinase with an effective amount of
one or more compounds disclosed herein. For instance, the step of
contacting involves the use of one or more compounds of any
formulae provided herein including but not limited to Formula I,
I-1, IV, IV-A, V, V-A, V-A2, V-B, VI, and VI-A. In some
embodiments, the step of contacting comprises contacting a cell
that contains said PI3 kinase. In some embodiments of the method,
the inhibition takes place in a subject suffering from a disorder
associated with malfunctioning of one or more types of PI3 kinase.
Some exemplary diseases involving malfunctioning of one or more
types of PI3 kinases are selected from the group consisting of
autoimmune diseases, rheumatoid arthritis, respiratory disease,
allergic reactions, and various types of cancers. Where desired,
the compound used in the method has the structure of Formula IV,
wherein R.sup.11 is amino and R.sup.12 is substituted phenyl.
In some embodiments of the method, the inhibition takes place in a
subject suffering from rheumatoid arthritis or a respiratory
disease, and wherein the compound has the structure of Formula IV,
and wherein R.sup.11 is amino and R.sup.12 is bicyclic
heteroaryl.
In some embodiments, the method comprises administering a second
therapeutic agent to the subject.
In yet another aspect, the present invention provides a method of
treating a disease manifesting an undesired immune response. The
method comprises the step of administering to a subject in need
thereof, one or more compounds disclosed herein including compounds
of Formula I, I-1, IV, IV-A, V, V-A, V-B, VI, and/or VI-A, in an
amount that is effective in ameliorating said undesired immune
response. In some embodiments, the one or more compounds inhibit
T-cell independent B-cell activation as evidenced by a reduction in
production of anti-TNP IgG3 by at least about five folds when
administered in an amount less than about 30 mg/kg BID dose to a
test animal.
In some embodiments, the disease treated is associated with
swelling or pain of a joint of a subject. The method can be
effective in ameliorating one or more rheumatoid arthritis symptoms
as evidenced by reduction in mean joint diameter by at least about
10% after 17 days and/or reduction in ankle diameter by at least
5-10% or more after several days to weeks of treatment, including
for example reduction in ankle diameter by at least 5% after 7 days
of treatment. In another embodiment, the undesired immune response
is evidenced by enhanced production of anti-type II collagen
antibodies, and the use of one or more subject compounds reduces
the serum anti-type II collagen level at an ED50 of less than about
10 mg/kg.
INCORPORATION BY REFERENCE
All publications, patents, and patent applications mentioned in
this specification are herein incorporated by reference to the same
extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
FIG. 1 depicts an exemplary protocol for measuring T-cell
independent production of TNP specific antibodies in vivo.
FIG. 2 depicts the fold reduction in TNP specific IgG3 response to
antigens provided by compounds 7 and 53 of formula IV as compared
to a vehicle control, when administered orally.
FIG. 3 depicts the dose-dependent effect of twice daily oral
administration of compound 53 of formula IV in reducing the
increase in ankle diameter over time in a collagen-induced
developing arthritis model in rats. Also depicted are the results
from non-arthritic control rats, arthritic control rats
administered with a negative control vehicle, and arthritic control
rats treated twice daily with methotrexate.
FIG. 4 depicts the dose-dependent effect of compounds 7 and 53 of
formula W in improving ankle histopathology when administered in a
collagen-induced developing arthritis model in rats. Also depicted
are the results from arthritic control rats administered with
negative control vehicle or methotrexate.
FIG. 5 depicts the dose-dependent effect of compounds 7 and 53 of
formula W in improving knee histopathology when administered in a
collagen-induced developing arthritis model in rats. Also depicted
are the results from arthritic control rats administered with
negative control vehicle or positive control methotrexate.
FIG. 6 depicts the dose-dependent effect of compounds 7 and 53 of
formula W in reducing the level of anti-type II collagen antibodies
in vivo when administered to a collagen-induced developing
arthritis rat model. Also depicted are the results from arthritic
rats administered with negative control vehicle or
methotrexate.
FIG. 7 depicts the dose-dependent effect of compound 7 of formula
IV on improving ankle histopathology when administered in
collagen-induced developing arthritis model in rats. Also depicted
are the results from arthritic vehicle control rats and
methotrexate-treated arthritic rats.
FIG. 8 depicts the dose-dependent effect of compound 53 of formula
IV administered daily on ankle histopathology in a collagen-induced
established arthritis model in rats. Also depicted are the results
from arthritic vehicle control rats and Enbrel-treated arthritic
rats.
FIG. 9 depicts the dose-dependent effect of compound 53 of formula
IV administered twice daily on ankle histopathology in a
collagen-induced established arthritis model in rats. Also depicted
are the results from arthritic vehicle control rats and
Enbrel-treated arthritic rats.
FIG. 10 depicts the dose-dependent effect of compound 53 of formula
IV on the increase in average paw volume in an adjuvant induced
arthritis model.
FIG. 11 depicts the effect of compound 53 of formula IV on the
average weight over time of rats in an adjuvant induced arthritis
model in rats.
DETAILED DESCRIPTION OF THE INVENTION
While preferred embodiments of the present invention have been
shown and described herein, it will be obvious to those skilled in
the art that such embodiments are provided by way of example only.
Numerous variations, changes, and substitutions will now occur to
those skilled in the art without departing from the invention. It
should be understood that various alternatives to the embodiments
of the invention described herein may be employed in practicing the
invention. It is intended that the appended claims define the scope
of the invention and that methods and structures within the scope
of these claims and their equivalents be covered thereby.
Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as is commonly understood by one of
skill in the art to which this invention belongs. All patents and
publications referred to herein are incorporated by reference.
As used in the specification and claims, the singular form "a",
"an" and "the" includes plural references unless the context
clearly dictates otherwise.
As used herein, "agent" or "biologically active agent" refers to a
biological, pharmaceutical, or chemical compound or other moiety.
Non-limiting examples include simple or complex organic or
inorganic molecule, a peptide, a protein, an oligonucleotide, an
antibody, an antibody derivative, antibody fragment, a vitamin
derivative, a carbohydrate, a toxin, or a chemotherapeutic
compound. Various compounds can be synthesized, for example, small
molecules and oligomers (e.g., oligopeptides and oligonucleotides),
and synthetic organic compounds based on various core structures.
In addition, various natural sources can provide compounds for
screening, such as plant or animal extracts, and the like. A
skilled artisan can readily recognize that there is no limit as to
the structural nature of the agents of the present invention.
The term "agonist" as used herein refers to a compound having the
ability to initiate or enhance a biological function of a target
protein, whether by inhibiting the activity or expression of the
target protein. Accordingly, the term "agonist" is defined in the
context of the biological role of the target polypeptide. While
preferred agonists herein specifically interact with (e.g. bind to)
the target, compounds that initiate or enhance a biological
activity of the target polypeptide by interacting with other
members of the signal transduction pathway of which the target
polypeptide is a member are also specifically included within this
definition.
The terms "antagonist" and "inhibitor" are used interchangeably,
and they refer to a compound having the ability to inhibit a
biological function of a target protein, whether by inhibiting the
activity or expression of the target protein. Accordingly, the
terms "antagonist" and "inhibitors" are defined in the context of
the biological role of the target protein. While preferred
antagonists herein specifically interact with (e.g. bind to) the
target, compounds that inhibit a biological activity of the target
protein by interacting with other members of the signal
transduction pathway of which the target protein is a member are
also specifically included within this definition. A preferred
biological activity inhibited by an antagonist is associated with
the development, growth, or spread of a tumor, or an undesired
immune response as manifested in autoimmune disease.
An "anti-cancer agent", "anti-tumor agent" or "chemotherapeutic
agent" refers to any agent useful in the treatment of a neoplastic
condition. One class of anti-cancer agents comprises
chemotherapeutic agents. "Chemotherapy" means the administration of
one or more chemotherapeutic drugs and/or other agents to a cancer
patient by various methods, including intravenous, oral,
intramuscular, intraperitoneal, intravesical, subcutaneous,
transdermal, buccal, or inhalation or in the form of a
suppository.
The term "cell proliferation" refers to a phenomenon by which the
cell number has changed as a result of division. This term also
encompasses cell growth by which the cell morphology has changed
(e.g., increased in size) consistent with a proliferative
signal.
The term "co-administration," "administered in combination with,"
and their grammatical equivalents, as used herein, encompass
administration of two or more agents to an animal so that both
agents and/or their metabolites are present in the animal at the
same time. Co-administration includes simultaneous administration
in separate compositions, administration at different times in
separate compositions, or administration in a composition in which
both agents are present.
The term "effective amount" or "therapeutically effective amount"
refers to that amount of a compound described herein that is
sufficient to effect the intended application including but not
limited to disease treatment, as defined below. The therapeutically
effective amount may vary depending upon the intended application
(in vitro or in vivo), or the subject and disease condition being
treated, e.g., the weight and age of the subject, the severity of
the disease condition, the manner of administration and the like,
which can readily be determined by one of ordinary skill in the
art. The term also applies to a dose that will induce a particular
response in target cells, e.g. reduction of platelet adhesion
and/or cell migration. The specific dose will vary depending on the
particular compounds chosen, the dosing regimen to be followed,
whether it is administered in combination with other compounds,
timing of administration, the tissue to which it is administered,
and the physical delivery system in which it is carried.
As used herein, "treatment" or "treating," or "palliating" or
"ameliorating" is used interchangeably herein. These terms refer to
an approach for obtaining beneficial or desired results including
but not limited to therapeutic benefit and/or a prophylactic
benefit. By therapeutic benefit is meant eradication or
amelioration of the underlying disorder being treated. Also, a
therapeutic benefit is achieved with the eradication or
amelioration of one or more of the physiological symptoms
associated with the underlying disorder such that an improvement is
observed in the patient, notwithstanding that the patient may still
be afflicted with the underlying disorder. For prophylactic
benefit, the compositions may be administered to a patient at risk
of developing a particular disease, or to a patient reporting one
or more of the physiological symptoms of a disease, even though a
diagnosis of this disease may not have been made.
A "therapeutic effect," as that term is used herein, encompasses a
therapeutic benefit and/or a prophylactic benefit as described
above. A prophylactic effect includes delaying or eliminating the
appearance of a disease or condition, delaying or eliminating the
onset of symptoms of a disease or condition, slowing, halting, or
reversing the progression of a disease or condition, or any
combination thereof.
The term "pharmaceutically acceptable salt" refers to salts derived
from a variety of organic and inorganic counter ions well known in
the art. Pharmaceutically acceptable acid addition salts can be
formed with inorganic acids and organic acids. Inorganic acids from
which salts can be derived include, for example, hydrochloric acid,
hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and
the like. Organic acids from which salts can be derived include,
for example, acetic acid, propionic acid, glycolic acid, pyruvic
acid, oxalic acid, maleic acid, malonic acid, succinic acid,
fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic
acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,
p-toluenesulfonic acid, salicylic acid, and the like.
Pharmaceutically acceptable base addition salts can be formed with
inorganic and organic bases. Inorganic bases from which salts can
be derived include, for example, sodium, potassium, lithium,
ammonium, calcium, magnesium, iron, zinc, copper, manganese,
aluminum, and the like. Organic bases from which salts can be
derived include, for example, primary, secondary, and tertiary
amines, substituted amines including naturally occurring
substituted amines, cyclic amines, basic ion exchange resins, and
the like, specifically such as isopropylamine, trimethylamine,
diethylamine, triethylamine, tripropylamine, and ethanolamine. In
some embodiments, the pharmaceutically acceptable base addition
salt is chosen from ammonium, potassium, sodium, calcium, and
magnesium salts.
"Pharmaceutically acceptable carrier" or "pharmaceutically
acceptable excipient" includes any and all solvents, dispersion
media, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents and the like. The use of such media and
agents for pharmaceutically active substances is well known in the
art. Except insofar as any conventional media or agent is
incompatible with the active ingredient, its use in the therapeutic
compositions of the invention is contemplated. Supplementary active
ingredients can also be incorporated into the compositions.
"Signal transduction" is a process during which stimulatory or
inhibitory signals are transmitted into and within a cell to elicit
an intracellular response. A modulator of a signal transduction
pathway refers to a compound which modulates the activity of one or
more cellular proteins mapped to the same specific signal
transduction pathway. A modulator may augment (agonist) or suppress
(antagonist) the activity of a signaling molecule.
The term "selective inhibition" or "selectively inhibit" as applied
to a biologically active agent refers to the agent's ability to
selectively reduce the target signaling activity as compared to
off-target signaling activity, via direct or interact interaction
with the target.
The term "B-ALL" as used herein refers to B-cell Acute
Lymphoblastic Leukemia.
"Subject" refers to an animal, such as a mammal, for example a
human. The methods described herein can be useful in both human
therapeutics and veterinary applications. In some embodiments, the
patient is a mammal, and in some embodiments, the patient is
human.
"Radiation therapy" means exposing a patient, using routine methods
and compositions known to the practitioner, to radiation emitters
such as alpha-particle emitting radionucleotides (e.g., actinium
and thorium radionuclides), low linear energy transfer (LET)
radiation emitters (i.e. beta emitters), conversion electron
emitters (e.g. strontium-89 and samarium-153-EDTMP, or high-energy
radiation, including without limitation x-rays, gamma rays, and
neutrons.
"Prodrug" is meant to indicate a compound that may be converted
under physiological conditions or by solvolysis to a biologically
active compound described herein. Thus, the term "prodrug" refers
to a precursor of a biologically active compound that is
pharmaceutically acceptable. A prodrug may be inactive when
administered to a subject, but is converted in vivo to an active
compound, for example, by hydrolysis. The prodrug compound often
offers advantages of solubility, tissue compatibility or delayed
release in a mammalian organism (see, e.g., Bundgard, H., Design of
Prodrugs (1985), pp. 7-9, 21-24 (Elsevier, Amsterdam). A discussion
of prodrugs is provided in Higuchi, T., et al., "Pro-drugs as Novel
Delivery Systems," A.C.S. Symposium Series, Vol. 14, and in
Bioreversible Carriers in Drug Design, ed. Edward B. Roche,
American Pharmaceutical Association and Pergamon Press, 1987, both
of which are incorporated in full by reference herein. The term
"prodrug" is also meant to include any covalently bonded carriers,
which release the active compound in vivo when such prodrug is
administered to a mammalian subject. Prodrugs of an active
compound, as described herein, may be prepared by modifying
functional groups present in the active compound in such a way that
the modifications are cleaved, either in routine manipulation or in
vivo, to the parent active compound. Prodrugs include compounds
wherein a hydroxy, amino or mercapto group is bonded to any group
that, when the prodrug of the active compound is administered to a
mammalian subject, cleaves to form a free hydroxy, free amino or
free mercapto group, respectively. Examples of prodrugs include,
but are not limited to, acetate, formate and benzoate derivatives
of an alcohol or acetamide, formamide and benzamide derivatives of
an amine functional group in the active compound and the like.
The term "in vivo" refers to an event that takes place in a
subject's body.
The term "in vitro" refers to an event that takes places outside of
a subject's body. For example, an in vitro assay encompasses any
assay run outside of a subject assay. In vitro assays encompass
cell-based assays in which cells alive or dead are employed. In
vitro assays also encompass a cell-free assay in which no intact
cells are employed.
Unless otherwise stated, structures depicted herein are also meant
to include compounds which differ only in the presence of one or
more isotopically enriched atoms. For example, compounds having the
present structures wherein hydrogen is replaced by deuterium or
tritium, or wherein carbon atom is replaced by .sup.13C- or
.sup.14C-enriched carbon, are within the scope of this
invention.
The compounds of the present invention may also contain unnatural
proportions of atomic isotopes at one or more of atoms that
constitute such compounds. For example, the compounds may be
radiolabeled with radioactive isotopes, such as for example tritium
(.sup.3H), iodine-125 (.sup.125I) or carbon-14 (.sup.14C). All
isotopic variations of the compounds of the present invention,
whether radioactive or not, are encompassed within the scope of the
present invention.
When ranges are used herein for physical properties, such as
molecular weight, or chemical properties, such as chemical
formulae, all combinations and subcombinations of ranges and
specific embodiments therein are intended to be included. The term
"about" when referring to a number or a numerical range means that
the number or numerical range referred to is an approximation
within experimental variability (or within statistical experimental
error), and thus the number or numerical range may vary from, for
example, between 1% and 15% of the stated number or numerical
range. The term "comprising" (and related terms such as "comprise"
or "comprises" or "having" or "including") includes those
embodiments, for example, an embodiment of any composition of
matter, composition, method, or process, or the like, that "consist
of" or "consist essentially of" the described features.
The following abbreviations and terms have the indicated meanings
throughout PI3-K=Phosphoinositide 3-kinase;
PI=phosphatidylinositol; PDK=Phosphoinositide Dependent Kinase;
DNA-PK=Deoxyribose Nucleic Acid Dependent Protein Kinase;
PTEN=Phosphatase and Tensin homolog deleted on chromosome Ten;
PIKK=Phosphoinositide Kinase Like Kinase; AIDS=Acquired Immuno
Deficiency Syndrome; HIV=Human Immunodeficiency Virus; MeI=Methyl
Iodide; POCl.sub.3=Phosphorous Oxychloride; KCNS=Potassium
IsoThiocyanate; TLC=Thin Layer Chromatography; MeOH=Methanol; and
CHCl.sub.3=Chloroform.
Abbreviations used herein have their conventional meaning within
the chemical and biological arts.
"Alkyl" refers to a straight or branched hydrocarbon chain radical
consisting solely of carbon and hydrogen atoms, containing no
unsaturation, having from one to ten carbon atoms (e.g.,
C.sub.1-C.sub.10 alkyl). Whenever it appears herein, a numerical
range such as "1 to 10" refers to each integer in the given range;
e.g., "1 to 10 carbon atoms" means that the alkyl group may consist
of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and
including 10 carbon atoms, although the present definition also
covers the occurrence of the term "alkyl" where no numerical range
is designated. In some embodiments, it is a C.sub.1-C.sub.4 alkyl
group. Typical alkyl groups include, but are in no way limited to,
methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl
isobutyl, tertiary butyl, pentyl, isopentyl, neopentyl, hexyl,
septyl, octyl, nonyl, decyl, and the like. The alkyl is attached to
the rest of the molecule by a single bond, for example, methyl
(Me), ethyl (Et), n-propyl, 1-methylethyl(iso-propyl), n-butyl,
n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl,
2-methylhexyl, and the like. Unless stated otherwise specifically
in the specification, an alkyl group is optionally substituted by
one or more of substituents which independently are: alkyl,
heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl,
arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano,
trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl,
--OR.sup.a, --SR.sup.a, --OC(O)--R.sup.a, --N(R.sub.a).sub.2,
--C(O)R.sup.a, --C(O)OR.sup.a, --OC(O)N(R.sup.a).sub.2,
--C(O)N(R.sup.a).sub.2, --N(R.sup.a)C(O)OR.sup.a,
--N(R.sup.a)C(O)R.sup.a, --N(R.sup.a)C(O)N(R.sup.a).sub.2,
N(R.sup.a)C(NR.sup.a)N(R.sup.a).sub.2,
--N(R.sup.a)S(O).sub.tR.sup.a (where t is 1 or 2),
--S(O).sub.tOR.sup.a (where t is 1 or 2),
--S(O).sub.tN(R.sup.a).sub.2 (where t is 1 or 2), or
PO.sub.3(R.sup.a).sub.2 where each R.sup.a is independently
hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl,
aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or
heteroarylalkyl.
"Alkylaryl" refers to an -(alkyl)aryl radical where aryl and alkyl
are as disclosed herein and which are optionally substituted by one
or more of the substituents described as suitable substituents for
aryl and alkyl respectively.
"Alkylhetaryl" refers to an -(alkyl)hetaryl radical where hetaryl
and alkyl are as disclosed herein and which are optionally
substituted by one or more of the substituents described as
suitable substituents for aryl and alkyl respectively.
"Alkylheterocycloalkyl" refers to an -(alkyl) heterocycyl radical
where alkyl and heterocycloalkyl are as disclosed herein and which
are optionally substituted by one or more of the substituents
described as suitable substituents for heterocycloalkyl and alkyl
respectively.
An "alkene" moiety refers to a group consisting of at least two
carbon atoms and at least one carbon-carbon double bond, and an
"alkyne" moiety refers to a group consisting of at least two carbon
atoms and at least one carbon-carbon triple bond. The alkyl moiety,
whether saturated or unsaturated, may be branched, straight chain,
or cyclic.
"Alkenyl" refers to a straight or branched hydrocarbon chain
radical group consisting solely of carbon and hydrogen atoms,
containing at least one double bond, and having from two to ten
carbon atoms (ie. C.sub.2-C.sub.10 alkenyl). Whenever it appears
herein, a numerical range such as "2 to 10" refers to each integer
in the given range; e.g., "2 to 10 carbon atoms" means that the
alkenyl group may consist of 2 carbon atoms, 3 carbon atoms, etc.,
up to and including 10 carbon atoms. In certain embodiments, an
alkenyl comprises two to eight carbon atoms. In other embodiments,
an alkenyl comprises two to five carbon atoms (e.g.,
C.sub.2-C.sub.5 alkenyl). The alkenyl is attached to the rest of
the molecule by a single bond, for example, ethenyl (i.e., vinyl),
prop-1-enyl (i.e., allyl), but-1-enyl, pent-1-enyl,
penta-1,4-dienyl, and the like. Unless stated otherwise
specifically in the specification, an alkenyl group is optionally
substituted by one or more substituents which independently are:
alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,
aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano,
trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl,
--OR.sup.a, --SR.sup.a, --OC(O)--R.sup.a, --N(R.sup.a).sub.2,
--C(O)R.sup.a, --C(O)OR.sup.a, --OC(O)N(R.sup.a).sub.2,
--C(O)N(R.sup.a).sub.2, --N(R.sup.a)C(O)OR.sup.a,
--N(R.sup.a)C(O)R.sup.a, --N(R.sup.a)C(O)N(R.sup.a).sub.2,
N(R.sup.a)C(NR.sup.a)N(R.sup.a).sub.2,
--N(R.sup.a)S(O).sub.tR.sup.a (where t is 1 or 2),
--S(O).sub.tOR.sup.a (where t is 1 or 2),
--S(O).sub.tN(R.sup.a).sub.2 (where t is 1 or 2), or
PO.sub.3(R.sup.a).sub.2, where each R.sup.a is independently
hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl,
aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or
heteroarylalkyl.
"Alkenyl-cycloalkyl" refers to an -(alkenyl)cycloalkyl radical
where alkenyl and cyclo alkyl are as disclosed herein and which are
optionally substituted by one or more of the substituents described
as suitable substituents for alkenyl and cycloalkyl
respectively.
"Alkynyl" refers to a straight or branched hydrocarbon chain
radical group consisting solely of carbon and hydrogen atoms,
containing at least one triple bond, having from two to ten carbon
atoms (ie. C.sub.2-C.sub.10 alkynyl). Whenever it appears herein, a
numerical range such as "2 to 10" refers to each integer in the
given range; e.g., "2 to 10 carbon atoms" means that the alkynyl
group may consist of 2 carbon atoms, 3 carbon atoms, etc., up to
and including 10 carbon atoms. In certain embodiments, an alkynyl
comprises two to eight carbon atoms. In other embodiments, an
alkynyl has two to five carbon atoms (e.g., C.sub.2-C.sub.5
alkynyl). The alkynyl is attached to the rest of the molecule by a
single bond, for example, ethynyl, propynyl, butynyl, pentynyl,
hexynyl, and the like. Unless stated otherwise specifically in the
specification, an alkynyl group is optionally substituted by one or
more substituents which independently are: alkyl, heteroalkyl,
alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl,
heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl,
trifluoromethoxy, nitro, trimethylsilanyl, --OR.sup.a, --SR.sup.a,
--OC(O)--R.sup.a, --N(R.sup.a).sub.2, --C(O)R.sup.a,
--C(O)OR.sup.a, --OC(O)N(R.sup.a).sub.2, --C(O)N(R.sup.a).sub.2,
--N(R.sup.a)C(O)OR.sup.a, --N(R.sup.a)C(O)R.sup.a,
--N(R.sup.a)C(O)N(R.sup.a).sub.2,
N(R.sup.a)C(NR.sup.a)N(R.sup.a).sub.2,
--N(R.sup.a)S(O).sub.tR.sup.a (where t is 1 or 2),
--S(O).sub.tOR.sup.a (where t is 1 or 2),
--S(O).sub.tN(R.sup.a).sub.2 (where t is 1 or 2), or
PO.sub.3(R.sup.a).sub.2, where each R.sup.a is independently
hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl,
aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or
heteroarylalkyl.
"Alkynyl-cycloalkyl" refers to an -(alkynyl)cycloalkyl radical
where alkynyl and cyclo alkyl are as disclosed herein and which are
optionally substituted by one or more of the substituents described
as suitable substituents for alkynyl and cycloalkyl
respectively.
"Carboxaldehyde" refers to a --(C.dbd.O)H radical.
"Carboxyl" refers to a --(C.dbd.O)OH radical.
"Cyano" refers to a --CN radical.
"Cycloalkyl" refers to a monocyclic or polycyclic radical that
contains only carbon and hydrogen, and may be saturated, or
partially unsaturated. Cycloalkyl groups include groups having from
3 to 10 ring atoms (ie. C.sub.2-C.sub.10 cycloalkyl). Whenever it
appears herein, a numerical range such as "3 to 10" refers to each
integer in the given range; e.g., "3 to 10 carbon atoms" means that
the cycloalkyl group may consist of 3 carbon atoms, etc., up to and
including 10 carbon atoms. In some embodiments, it is a
C.sub.3-C.sub.8 cycloalkyl radical. In some embodiments, it is a
C.sub.3-C.sub.5 cycloalkyl radical. Illustrative examples of
cycloalkyl groups include, but are not limited to the following
moieties: cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl,
cyclohexyl, cyclohexenyl, cycloseptyl, cyclooctyl, cyclononyl,
cyclodecyl, norbornyl, and the like. Unless stated otherwise
specifically in the specification, a cycloalkyl group is optionally
substituted by one or more substituents which independently are:
alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,
aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano,
trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl,
--OR.sup.a, --SR.sup.a, --OC(O)--R.sup.a, --N(R.sup.a).sub.2,
--C(O)R.sup.a, --C(O)OR.sup.a, --OC(O)N(R.sup.a).sub.2,
--C(O)N(R.sup.a).sub.2, --N(R.sup.a)C(O)OR.sup.a,
--N(R.sup.a)C(O)R.sup.a, --N(R.sup.a)C(O)N(R.sup.a).sub.2,
N(R.sup.a)C(NR.sup.a)N(R.sup.a).sub.2,
--N(R.sup.a)S(O).sub.tR.sup.a (where t is 1 or 2),
--S(O).sub.tOR.sup.a (where t is 1 or 2),
--S(O).sub.tN(R.sup.a).sub.2 (where t is 1 or 2), or
PO.sub.3(R.sup.a).sub.2, where each R.sup.a is independently
hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl,
aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or
heteroarylalkyl.
"Cycloalkyl-alkenyl" refers to a -(cycloalkyl)alkenyl radical where
cycloalkyl and heterocycloalkyl are as disclosed herein and which
are optionally substituted by one or more of the substituents
described as suitable substituents for heterocycloalkyl and
cycloalkyl respectively.
"Cycloalkyl-heterocycloalkyl" refers to a -(cycloalkyl) heterocycyl
radical where cycloalkyl and heterocycloalkyl are as disclosed
herein and which are optionally substituted by one or more of the
substituents described as suitable substituents for
heterocycloalkyl and cycloalkyl respectively.
"Cycloalkyl-heteroaryl" refers to a -(cycloalkyl)heteroaryl radical
where cycloalkyl and heterocycloalkyl are as disclosed herein and
which are optionally substituted by one or more of the substituents
described as suitable substituents for heterocycloalkyl and
cycloalkyl respectively.
The term "alkoxy" refers to the group --O-alkyl, including from 1
to 8 carbon atoms of a straight, branched, cyclic configuration and
combinations thereof attached to the parent structure through an
oxygen. Examples include methoxy, ethoxy, propoxy, isopropoxy,
cyclopropyloxy, cyclohexyloxy and the like. "Lower alkoxy" refers
to alkoxy groups containing one to six carbons. In some
embodiments, C.sub.1-C.sub.4 alkyl, is an alkyl group which
encompasses both straight and branched chain alkyls of from 1 to 4
carbon atoms.
The term "substituted alkoxy" refers to alkoxy wherein the alkyl
constituent is substituted (i.e., --O-(substituted alkyl)). Unless
stated otherwise specifically in the specification, the alkyl
moiety of an alkoxy group is optionally substituted by one or more
substituents which independently are: alkyl, heteroalkyl, alkenyl,
alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,
heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl,
trifluoromethoxy, nitro, trimethylsilanyl, --OR.sup.a, SR.sup.a,
--OC(O)--R.sup.a, --N(R.sup.a).sub.2, --C(O)R.sup.a,
--C(O)OR.sup.a, --OC(O)N(R.sup.a).sub.2, --C(O)N(R.sup.a).sub.2,
--N(R.sup.a)C(O)OR.sup.a, --N(R.sup.a)C(O)R.sup.a,
--N(R.sup.a)C(O)N(R.sup.a).sub.2,
N(R.sup.a)C(NR.sup.a)N(R.sup.a).sub.2,
--N(R.sup.a)S(O).sub.tR.sup.a (where t is 1 or 2),
--S(O).sub.tOR.sup.a (where t is 1 or 2),
--S(O).sub.tN(R.sup.a).sub.2 (where t is 1 or 2), or
PO.sub.3(R.sup.a).sub.2, where each R.sup.a is independently
hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl,
aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or
heteroarylalkyl.
The term "alkoxycarbonyl" refers to a group of the formula
(alkoxy)(C.dbd.O)-- attached through the carbonyl carbon wherein
the alkoxy group has the indicated number of carbon atoms. Thus a
C.sub.1-C.sub.6 alkoxycarbonyl group is an alkoxy group having from
1 to 6 carbon atoms attached through its oxygen to a carbonyl
linker. "Lower alkoxycarbonyl" refers to an alkoxycarbonyl group
wherein the alkoxy group is a lower alkoxy group. In some
embodiments, C.sub.1-C.sub.4 alkoxy, is an alkoxy group which
encompasses both straight and branched chain alkoxy groups of from
1 to 4 carbon atoms.
The term "substituted alkoxycarbonyl" refers to the group
(substituted alkyl)-O--C(O)-- wherein the group is attached to the
parent structure through the carbonyl functionality. Unless stated
otherwise specifically in the specification, the alkyl moiety of an
alkoxycarbonyl group is optionally substituted by one or more
substituents which independently are: alkyl, heteroalkyl, alkenyl,
alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,
heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl,
trifluoromethoxy, nitro, trimethylsilanyl, --OR.sup.a, SR.sup.a,
--OC(O)--R.sup.a, --N(R.sup.a).sub.2, --C(O)R.sup.a,
--C(O)OR.sup.a, --OC(O)N(R.sup.3).sub.2, --C(O)N(R.sup.a).sub.2,
--N(R.sup.a)C(O)OR.sup.a, --N(R.sup.a)C(O)R.sup.a,
--N(R.sup.a)C(O)N(R.sup.a).sub.2,
N(R.sup.a)C(NR.sup.a)N(R.sup.a).sub.2,
--N(R.sup.a)S(O).sub.tR.sup.a (where t is 1 or 2),
--S(O).sub.tOR.sup.a (where t is 1 or 2),
--S(O).sub.tN(R.sup.a).sub.2 (where t is 1 or 2), or
PO.sub.3(R.sup.a).sub.2, where each R.sup.a is independently
hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl,
aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or
heteroarylalkyl.
"Acyl" refers to the groups (alkyl)-C(O)--, (aryl)-C(O)--,
(heteroaryl)-C(O)--, (heteroalkyl)-C(O)--, and
(heterocycloalkyl)-C(O)--, wherein the group is attached to the
parent structure through the carbonyl functionality. In some
embodiments, it is a C.sub.1-C.sub.10 acyl radical which refers to
the total number of chain or ring atoms of the alkyl, aryl,
heteroaryl or heterocycloalkyl portion of the acyloxy group plus
the carbonyl carbon of acyl, i.e three other ring or chain atoms
plus carbonyl. If the R radical is heteroaryl or heterocycloalkyl,
the hetero ring or chain atoms contribute to the total number of
chain or ring atoms. Unless stated otherwise specifically in the
specification, the "R" of an acyloxy group is optionally
substituted by one or more substituents which independently are:
alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,
aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano,
trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl,
--OR.sup.a, SR.sup.a, --OC(O)--R.sup.a, --N(R.sup.a).sub.2,
--C(O)R.sup.a, --C(O)OR.sup.a, --OC(O)N(R.sup.3).sub.2,
--C(O)N(R.sup.a).sub.2, --N(R.sup.a)C(O)OR.sup.a,
--N(R.sup.a)C(O)R.sup.a, --N(R.sup.a)C(O)N(R.sup.a).sub.2,
N(R.sup.a)C(NR.sup.a)N(R.sup.a).sub.2,
--N(R.sup.a)S(O).sub.tR.sup.a (where t is 1 or 2),
--S(O).sub.tOR.sup.a (where t is 1 or 2),
--S(O).sub.tN(R.sup.a).sub.2 (where t is 1 or 2), or
PO.sub.3(R.sup.a).sub.2, where each R.sup.a is independently
hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl,
aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or
heteroarylalkyl.
"Acyloxy" refers to a R(C.dbd.O)O-- radical wherein "R" is alkyl,
aryl, heteroaryl, heteroalkyl, or heterocycloalkyl, which are as
described herein. In some embodiments, it is a C.sub.1-C.sub.4
acyloxy radical which refers to the total number of chain or ring
atoms of the alkyl, aryl, heteroaryl or heterocycloalkyl portion of
the acyloxy group plus the carbonyl carbon of acyl, i.e three other
ring or chain atoms plus carbonyl. If the R radical is heteroaryl
or heterocycloalkyl, the hetero ring or chain atoms contribute to
the total number of chain or ring atoms. Unless stated otherwise
specifically in the specification, the "R" of an acyloxy group is
optionally substituted by one or more substituents which
independently are: alkyl, heteroalkyl, alkenyl, alkynyl,
cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,
heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl,
trifluoromethoxy, nitro, trimethylsilanyl, --OR.sup.a, --SR.sup.a,
--OC(O)--R.sup.a, --N(R.sup.a).sub.2, --C(O)R.sup.a,
--C(O)OR.sup.a, --OC(O)N(R.sup.a).sub.2, --C(O)N(R.sup.a).sub.2,
--N(R.sup.a)C(O)OR.sup.a, --N(R.sup.a)C(O)R.sup.a,
--N(R.sup.a)C(O)N(R.sup.a).sub.2,
N(R.sup.a)C(NR.sup.a)N(R.sup.a).sub.2,
--N(R.sup.a)S(O).sub.tR.sup.a (where t is 1 or 2-S(O).sub.tOR.sup.a
(where t is 1 or 2), --S(O).sub.tN(R.sup.a).sub.2 (where t is 1 or
2), or PO.sub.3(R.sup.a).sub.2, where each R.sup.a is independently
hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl,
aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or
heteroarylalkyl.
"Amino" or "amine" refers to a --N(R.sup.a).sub.2 radical group,
where each R.sup.a is independently hydrogen, alkyl, fluoroalkyl,
carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,
heterocycloalkylalkyl, heteroaryl or heteroarylalkyl, unless stated
otherwise specifically in the specification. When a
--N(R.sup.a).sub.2 group has two Ra other than hydrogen they can be
combined with the nitrogen atom to form a 4-, 5-, 6-, or 7-membered
ring. For example, --N(R.sup.a).sub.2 is meant to include, but not
be limited to, 1-pyrrolidinyl and 4-morpholinyl. Unless stated
otherwise specifically in the specification, an amino group is
optionally substituted by one or more substituents which
independently are: alkyl, heteroalkyl, alkenyl, alkynyl,
cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,
heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl,
trifluoromethoxy, nitro, trimethylsilanyl, --OR.sup.a, --SR.sup.a,
--OC(O)--R.sup.a, --N(R.sup.a).sub.2, --C(O)R.sup.a,
--C(O)OR.sup.a, --OC(O)N(R.sup.a).sub.2, --C(O)N(R.sup.a).sub.2,
--N(R.sup.a)C(O)OR.sup.a, --N(R.sup.a)C(O)R.sup.a,
--N(R.sup.a)C(O)N(R.sup.a).sub.2,
N(R.sup.a)C(NR.sup.a)N(R.sup.a).sub.2,
--N(R.sup.a)S(O).sub.tR.sup.a (where t is 1 or 2),
--S(O).sub.tOR.sup.a (where t is 1 or 2),
--S(O).sub.tN(R.sup.a).sub.2 (where t is 1 or 2), or
PO.sub.3(R.sup.a).sub.2, where each R.sup.a is independently
hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl,
aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or
heteroarylalkyl and each of these moieties may be optionally
substituted as defined herein.
The term "substituted amino" also refers to N-oxides of the groups
--NHR.sup.d, and NR.sup.dR.sup.d each as described above. N-oxides
can be prepared by treatment of the corresponding amino group with,
for example, hydrogen peroxide or m-chloroperoxybenzoic acid. The
person skilled in the art is familiar with reaction conditions for
carrying out the N-oxidation.
"Amide" or "amido" refers to a chemical moiety with formula
--C(O)N(R).sub.2 or --NHC(O)R, where R is selected from the group
consisting of hydrogen, alkyl, cycloalkyl, aryl, heteroaryl (bonded
through a ring carbon) and heteroalicyclic (bonded through a ring
carbon), each of which moiety may itself be optionally substituted.
In some embodiments it is a C.sub.1-C.sub.4 amido or amide radical,
which includes the amide carbonyl in the total number of carbons in
the radical. The R.sub.2 of --N(R).sub.2 of the amide may
optionally be taken together with the nitrogen to which it is
attached to form a 4-, 5-, 6-, or 7-membered ring. Unless stated
otherwise specifically in the specification, an amido group is
optionally substituted independently by one or more of the
substituents as described herein for alkyl, cycloalkyl, aryl,
heteroaryl, or heterocycloalkyl. An amide may be an amino acid or a
peptide molecule attached to a compound of Formula (I), thereby
forming a prodrug. Any amine, hydroxy, or carboxyl side chain on
the compounds described herein can be amidified. The procedures and
specific groups to make such amides are known to those of skill in
the art and can readily be found in reference sources such as
Greene and Wuts, Protective Groups in Organic Synthesis, 3.sup.rd
Ed., John Wiley & Sons, New York, N.Y., 1999, which is
incorporated herein by reference in its entirety.
"Aromatic" or "aryl" refers to an aromatic radical with six to ten
ring atoms (e.g., C.sub.6-C.sub.10 aromatic or C.sub.6-C.sub.10
aryl) which has at least one ring having a conjugated pi electron
system which is carbocyclic (e.g., phenyl, fluorenyl, and
naphthyl). Bivalent radicals formed from substituted benzene
derivatives and having the free valences at ring atoms are named as
substituted phenylene radicals. Bivalent radicals derived from
univalent polycyclic hydrocarbon radicals whose names end in "-yl"
by removal of one hydrogen atom from the carbon atom with the free
valence are named by adding "-idene" to the name of the
corresponding univalent radical, e.g., a naphthyl group with two
points of attachment is termed naphthylidene. Whenever it appears
herein, a numerical range such as "6 to 10" refers to each integer
in the given range; e.g., "6 to 10 ring atoms" means that the aryl
group may consist of 6 ring atoms, 7 ring atoms, etc., up to and
including 10 ring atoms. The term includes monocyclic or fused-ring
polycyclic (i.e., rings which share adjacent pairs of ring atoms)
groups. Unless stated otherwise specifically in the specification,
an aryl moiety is optionally substituted by one or more
substituents which are independently: alkyl, heteroalkyl, alkenyl,
alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,
heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl,
trifluoromethoxy, nitro, trimethylsilanyl, --OR.sup.a, --SR.sup.a,
--OC(O)--R.sup.a, --N(R.sup.a).sub.2, --C(O)R.sup.a,
--C(O)OR.sup.a, --OC(O)N(R.sup.a).sub.2, --C(O)N(R.sup.a).sub.2,
--N(R.sup.a)C(O)OR.sup.a, --N(R.sup.a)C(O)R.sup.a,
--N(R.sup.a)C(O)N(R.sup.a).sub.2,
N(R.sup.a)C(NR.sup.a)N(R.sup.a).sub.2,
--N(R.sup.a)S(O).sub.tR.sup.a (where t is 1 or 2),
--S(O).sub.tOR.sup.a (where t is 1 or 2),
--S(O).sub.tN(R.sup.a).sub.2 (where t is 1 or 2), or
PO.sub.3(R.sup.a).sub.2, where each R.sup.a is independently
hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl,
aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or
heteroarylalkyl.
"Aralkyl" or "arylalkyl" refers to an (aryl)alkyl-radical where
aryl and alkyl are as disclosed herein and which are optionally
substituted by one or more of the substituents described as
suitable substituents for aryl and alkyl respectively.
"Ester" refers to a chemical radical of formula --COOR, where R is
selected from the group consisting of alkyl, cycloalkyl, aryl,
heteroaryl (bonded through a ring carbon) and heteroalicyclic
(bonded through a ring carbon). Any amine, hydroxy, or carboxyl
side chain on the compounds described herein can be esterified. The
procedures and specific groups to make such esters are known to
those of skill in the art and can readily be found in reference
sources such as Greene and Wuts, Protective Groups in Organic
Synthesis, 3.sup.rd Ed., John Wiley & Sons, New York, N.Y.,
1999, which is incorporated herein by reference in its entirety.
Unless stated otherwise specifically in the specification, an ester
group is optionally substituted by one or more substituents which
independently are: alkyl, heteroalkyl, alkenyl, alkynyl,
cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,
heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl,
trifluoromethoxy, nitro, trimethylsilanyl, --OR.sup.a, --SR.sup.a,
--OC(O)--R.sup.a, --N(R.sup.a).sub.2, --C(O)R.sup.a,
--C(O)OR.sup.a, --OC(O)N(R.sup.a).sub.2, --C(O)N(R.sup.a).sub.2,
--N(R.sup.a)C(O)OR.sup.a, --N(R.sup.a)C(O)R.sup.a,
--N(R.sup.a)C(O)N(R.sup.a).sub.2,
N(R.sup.a)C(NR.sup.a)N(R.sup.a).sub.2,
--N(R.sup.a)S(O).sub.tR.sup.a (where t is 1 or 2),
--S(O).sub.tOR.sup.a (where t is 1 or 2),
--S(O).sub.tN(R.sup.a).sub.2 (where t is 1 or 2), or
PO.sub.3(R.sup.a).sub.2, where each R.sup.a is independently
hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl,
aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or
heteroarylalkyl.
"Fluoroalkyl" refers to an alkyl radical, as defined above, that is
substituted by one or more fluoro radicals, as defined above, for
example, trifluoromethyl, difluoromethyl, 2,2,2-trifluoroethyl,
1-fluoromethyl-2-fluoroethyl, and the like. The alkyl part of the
fluoroalkyl radical may be optionally substituted as defined above
for an alkyl group.
"Halo", "halide", or, alternatively, "halogen" means fluoro,
chloro, bromo or iodo. The terms "haloalkyl," "haloalkenyl,"
"haloalkynyl" and "haloalkoxy" include alkyl, alkenyl, alkynyl and
alkoxy structures that are substituted with one or more halo groups
or with combinations thereof. For example, the terms "fluoroalkyl"
and "fluoroalkoxy" include haloalkyl and haloalkoxy groups,
respectively, in which the halo is fluorine.
"Heteroalkyl" "heteroalkenyl" and "heteroalkynyl" include
optionally substituted alkyl, alkenyl and alkynyl radicals and
which have one or more skeletal chain atoms selected from an atom
other than carbon, e.g., oxygen, nitrogen, sulfur, phosphorus or
combinations thereof. A numerical range may be given, e.g.
C.sub.1-C.sub.4 heteroalkyl which refers to the chain length in
total, which in this example is 4 atoms long. For example, a
--CH.sub.2OCH.sub.2CH.sub.3 radical is referred to as a "C.sub.4"
heteroalkyl, which includes the heteroatom center in the atom chain
length description. Connection to the rest of the molecule may be
through either a heteroatom or a carbon in the heteroalkyl chain. A
heteroalkyl group may be substituted with one or more substituents
which independently are: alkyl, heteroalkyl, alkenyl, alkynyl,
cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,
heteroarylalkyl, hydroxy, halo, cyano, nitro, oxo, thioxo,
trimethylsilanyl, --OR.sup.a, --SR.sup.a, --OC(O)--R.sup.a,
--N(R.sup.a).sub.2, --C(O)R.sup.a, --C(O)OR.sup.a,
--C(O)N(R.sup.a).sub.2, --N(R.sup.a)C(O)OR.sup.a,
--N(R.sup.a)C(O)R.sup.a, --N(R.sup.a)S(O).sub.tR.sup.a (where t is
1 or 2), --S(O).sub.tOR.sup.a (where t is 1 or 2),
--S(O).sub.tN(R.sup.a).sub.2 (where t is 1 or 2), or
PO.sub.3(R.sup.a).sub.2, where each R.sup.a is independently
hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl,
aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or
heteroarylalkyl.
"Heteroalkylaryl" refers to an -(heteroalkyl)aryl radical where
heteroalkyl and aryl are as disclosed herein and which are
optionally substituted by one or more of the substituents described
as suitable substituents for heteroalkyl and aryl respectively.
"Heteroalkylheteroaryl" refers to an -(heteroalkyl)heteroaryl
radical where heteroalkyl and heteroaryl are as disclosed herein
and which are optionally substituted by one or more of the
substituents described as suitable substituents for heteroalkyl and
heteroaryl respectively.
"Heteroalkylheterocycloalkyl" refers to an
-(heteroalkyl)heterocycloalkyl radical where heteroalkyl and
heteroaryl are as disclosed herein and which are optionally
substituted by one or more of the substituents described as
suitable substituents for heteroalkyl and heterocycloalkyl
respectively.
"Heteroalkylcycloalkyl" refers to an -(heteroalkyl)cycloalkyl
radical where heteroalkyl and cycloalkyl are as disclosed herein
and which are optionally substituted by one or more of the
substituents described as suitable substituents for heteroalkyl and
cycloalkyl respectively.
"Heteroaryl" or, alternatively, "heteroaromatic" refers to a 5- to
18-membered aromatic radical (e.g., C.sub.5-C.sub.13 heteroaryl)
that includes one or more ring heteroatoms selected from nitrogen,
oxygen and sulfur, and which may be a monocyclic, bicyclic,
tricyclic or tetracyclic ring system. Whenever it appears herein, a
numerical range such as "5 to 18" refers to each integer in the
given range; e.g., "5 to 18 ring atoms" means that the heteroaryl
group may consist of 5 ring atoms, 6 ring atoms, etc., up to and
including 18 ring atoms. Bivalent radicals derived from univalent
heteroaryl radicals whose names end in "-yl" by removal of one
hydrogen atom from the atom with the free valence are named by
adding "-idene" to the name of the corresponding univalent radical,
e.g., a pyridyl group with two points of attachment is a
pyridylidene. An N-containing "heteroaromatic" or "heteroaryl"
moiety refers to an aromatic group in which at least one of the
skeletal atoms of the ring is a nitrogen atom. The polycyclic
heteroaryl group may be fused or non-fused. The heteroatom(s) in
the heteroaryl radical is optionally oxidized. One or more nitrogen
atoms, if present, are optionally quaternized. The heteroaryl is
attached to the rest of the molecule through any atom of the
ring(s). Examples of heteroaryls include, but are not limited to,
azepinyl, acridinyl, benzimidazolyl, benzindolyl,
1,3-benzodioxolyl, benzofuranyl, benzooxazolyl, benzo[d]thiazolyl,
benzothiadiazolyl, benzo[b][1,4]dioxepinyl, benzo[b][1,4]oxazinyl,
1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl,
benzodioxolyl, benzodioxinyl, benzoxazolyl, benzopyranyl,
benzopyranonyl, benzofuranyl, benzopyranonyl, benzofurazanyl,
benzothiazolyl, benzothienyl(benzothiophenyl),
benzothieno[3,2-d]pyrimidinyl, benzotriazolyl,
benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl,
cyclopenta[d]pyrimidinyl,
6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidinyl,
5,6-dihydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]cinnolinyl,
6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazinyl,
dibenzofuranyl, dibenzothiophenyl, furanyl, furazanyl, furanonyl,
furo[3,2-c]pyridinyl,
5,6,7,8,9,10-hexahydrocycloocta[d]pyrimidinyl,
5,6,7,8,9,10-hexahydrocycloocta[d]pyridazinyl,
5,6,7,8,9,10-hexahydrocycloocta[d]pyridinyl, isothiazolyl,
imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl,
isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl,
5,8-methano-5,6,7,8-tetrahydroquinazolinyl, naphthyridinyl,
1,6-naphthyridinonyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl,
oxiranyl, 5,6,6a,7,8,9,10,10a-octahydrobenzo[h]quinazolinyl,
1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl,
phthalazinyl, pteridinyl, purinyl, pyranyl, pyrrolyl, pyrazolyl,
pyrazolo[3,4-d]pyrimidinyl, pyridinyl, pyrido[3,2-d]pyrimidinyl,
pyrido[3,4-d]pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl,
pyrrolyl, quinazolinyl, quinoxalinyl, quinolinyl, isoquinolinyl,
tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl,
5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl,
6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidinyl,
5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl, thiazolyl,
thiadiazolyl, thiapyranyl, triazolyl, tetrazolyl, triazinyl,
thieno[2,3-d]pyrimidinyl, thieno[3,2-d]pyrimidinyl,
thieno[2,3-c]pridinyl, and thiophenyl (i.e. thienyl). Unless stated
otherwise specifically in the specification, a heteraryl moiety is
optionally substituted by one or more substituents which are
independently: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl,
heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl,
hydroxy, halo, cyano, nitro, oxo, thioxo, trimethylsilanyl,
--OR.sup.a, --SR.sup.a, --OC(O)--R.sup.a, --N(R.sup.a).sub.2,
--C(O)R.sup.a, --C(O)OR.sup.a, --C(O)N(R.sup.a).sub.2,
--N(R.sup.a)C(O)OR.sup.a, --N(R.sup.a)C(O)R.sup.a,
--N(R.sup.a)S(O).sub.tR.sup.a (where t is 1 or 2),
--S(O).sub.tOR.sup.a (where t is 1 or 2),
--S(O).sub.tN(R.sup.a).sub.2 (where t is 1 or 2), or
PO.sub.3(R.sup.a).sub.2, where each R.sup.a is independently
hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl,
aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or
heteroarylalkyl.
Substituted heteroaryl also includes ring systems substituted with
one or more oxide (--O--) substituents, such as pyridinyl
N-oxides.
"Heteroarylalkyl" refers to a moiety having an aryl moiety, as
described herein, connected to an alkylene moiety, as described
herein, wherein the connection to the remainder of the molecule is
through the alkylene group.
"Heterocycloalkyl" refers to a stable 3- to 18-membered
non-aromatic ring radical that comprises two to twelve carbon atoms
and from one to six heteroatoms selected from nitrogen, oxygen and
sulfur. Whenever it appears herein, a numerical range such as "3 to
18" refers to each integer in the given range; e.g., "3 to 18 ring
atoms" means that the heterocycloalkyl group may consist of 3 ring
atoms, 4 ring atoms, etc., up to and including 18 ring atoms. In
some embodiments, it is a C.sub.5-C.sub.10 heterocycloalkyl. In
some embodiments, it is a C.sub.4-C.sub.10 heterocycloalkyl. In
some embodiments, it is a C.sub.3-C.sub.10 heterocycloalkyl. Unless
stated otherwise specifically in the specification, the
heterocycloalkyl radical is a monocyclic, bicyclic, tricyclic or
tetracyclic ring system, which may include fused or bridged ring
systems. The heteroatoms in the heterocycloalkyl radical may be
optionally oxidized. One or more nitrogen atoms, if present, are
optionally quaternized. The heterocycloalkyl radical is partially
or fully saturated. The heterocycloalkyl may be attached to the
rest of the molecule through any atom of the ring(s). Examples of
such heterocycloalkyl radicals include, but are not limited to,
dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl,
imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl,
morpholinyl, octahydroindolyl, octahydroisoindolyl,
2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl,
oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl,
pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl,
tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl,
thiamorpholinyl, 1-oxo-thiomorpholinyl, and
1,1-dioxo-thiomorpholinyl. Unless stated otherwise specifically in
the specification, a heterocycloalkyl moiety is optionally
substituted by one or more substituents which independently are:
alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,
aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano,
nitro, oxo, thioxo, trimethylsilanyl, --OR.sup.a, --SR.sup.a,
--OC(O)--R.sup.a, --N(R.sup.a).sub.2, --C(O)R.sup.a,
--C(O)OR.sup.a, --C(O)N(R.sup.a).sub.2, --N(R.sup.a)C(O)OR.sup.a,
--N(R.sup.a)C(O)R.sup.a, --N(R.sup.a)S(O).sub.tR.sup.a (where t is
1 or 2), --S(O).sub.tOR.sup.a (where t is 1 or 2),
--S(O).sub.tN(R.sup.a).sub.2 (where t is 1 or 2), or
PO.sub.3(R.sup.a).sub.2, where each R.sup.a is independently
hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl,
aralkyl, heterocycloalkyl, heteroaryl or heteroarylalkyl.
"Heterocycloalkyl" also includes bicyclic ring systems wherein one
non-aromatic ring, usually with 3 to 7 ring atoms, contains at
least 2 carbon atoms in addition to 1-3 heteroatoms independently
selected from oxygen, sulfur, and nitrogen, as well as combinations
comprising at least one of the foregoing heteroatoms; and the other
ring, usually with 3 to 7 ring atoms, optionally contains 1-3
heteroatoms independently selected from oxygen, sulfur, and
nitrogen and is not aromatic.
"Isomers" are different compounds that have the same molecular
formula. "Stereoisomers" are isomers that differ only in the way
the atoms are arranged in space, i.e. having a different
stereochemical configuration. "Enantiomers" are a pair of
stereoisomers that are non-superimposable mirror images of each
other. A 1:1 mixture of a pair of enantiomers is a "racemic"
mixture. The term "(..+-..)" is used to designate a racemic mixture
where appropriate. "Diastereoisomers" are stereoisomers that have
at least two asymmetric atoms, but which are not mirror-images of
each other. The absolute stereochemistry is specified according to
the Cahn-Ingold-Prelog R-S system. When a compound is a pure
enantiomer the stereochemistry at each chiral carbon can be
specified by either R or S. Resolved compounds whose absolute
configuration is unknown can be designated (+) or (-) depending on
the direction (dextro- or levorotatory) which they rotate plane
polarized light at the wavelength of the sodium D line. Certain of
the compounds described herein contain one or more asymmetric
centers and can thus give rise to enantiomers, diastereomers, and
other stereoisomeric forms that can be defined, in terms of
absolute stereochemistry, as (R)- or (S)-. The present chemical
entities, pharmaceutical compositions and methods are meant to
include all such possible isomers, including racemic mixtures,
optically pure forms and intermediate mixtures. Optically active
(R)- and (S)-isomers can be prepared using chiral synthons or
chiral reagents, or resolved using conventional techniques. When
the compounds described herein contain olefinic double bonds or
other centers of geometric asymmetry, and unless specified
otherwise, it is intended that the compounds include both E and Z
geometric isomers.
"Enantiomeric purity" as used herein refers to the relative
amounts, expressed as a percentage, of the presence of a specific
enantiomer relative to the other enantiomer. For example, if a
compound, which may potentially have an (R)- or an (S)-isomeric
configuration, is present as a racemic mixture, the enantiomeric
purity is about 50% with respect to either the (R)- or (S)-isomer.
If that compound has one isomeric form predominant over the other,
for example, 80% (S)- and 20% (R)-, the enantiomeric purity of the
compound with respect to the (S)-isomeric form is 80%. The
enantiomeric purity of a compound can be determined in a number of
ways known in the art, including but not limited to chromatography
using a chiral support, polarimetric measurement of the rotation of
polarized light, nuclear magnetic resonance spectroscopy using
chiral shift reagents which include but are not limited to
lanthanide containing chiral complexes or the Pirkle alcohol, or
derivatization of a compounds using a chiral compound such as
Mosher's acid followed by chromatography or nuclear magnetic
resonance spectroscopy.
"Moiety" refers to a specific segment or functional group of a
molecule. Chemical moieties are often recognized chemical entities
embedded in or appended to a molecule.
"Nitro" refers to the NO.sub.2 radical.
"Oxa" refers to the --O-- radical.
"Oxo" refers to the .dbd.O radical.
"Tautomers" are structurally distinct isomers that interconvert by
tautomerization. "Tautomerization" is a form of isomerization and
includes prototropic or proton-shift tautomerization, which is
considered a subset of acid-base chemistry. "Prototropic
tautomerization" or "proton-shift tautomerization" involves the
migration of a proton accompanied by changes in bond order, often
the interchange of a single bond with an adjacent double bond.
Where tautomerization is possible (e.g. in solution), a chemical
equilibrium of tautomers can be reached. An example of
tautomerization is keto-enol tautomerization. A specific example of
keto-enol tautomerization is the interconversion of
pentane-2,4-dione and 4-hydroxypent-3-en-2-one tautomers. Another
example of tautomerization is phenol-keto tautomerization. A
specific example of phenol-keto tautomerization is the
interconversion of pyridin-4-ol and pyridin-4(1H)-one
tautomers.
The compounds of the present invention may also contain unnatural
proportions of atomic isotopes at one or more of atoms that
constitute such compounds. For example, the compounds may be
radiolabeled with radioactive isotopes, such as for example tritium
(.sup.3H), iodine-125 (.sup.125I) or carbon-14 (.sup.14C). All
isotopic variations of the compounds of the present invention,
whether radioactive or not, are encompassed within the scope of the
present invention.
A "leaving group or atom" is any group or atom that will, under the
reaction conditions, cleave from the starting material, thus
promoting reaction at a specified site. Suitable examples of such
groups unless otherwise specified are halogen atoms, mesyloxy,
p-nitrobenzensulphonyloxy and tosyloxy groups.
"Protecting group" has the meaning conventionally associated with
it in organic synthesis, i.e. a group that selectively blocks one
or more reactive sites in a multifunctional compound such that a
chemical reaction can be carried out selectively on another
unprotected reactive site and such that the group can readily be
removed after the selective reaction is complete. A variety of
protecting groups are disclosed, for example, in T. H. Greene and
P. G. M. Wuts, Protective Groups in Organic Synthesis, Third
Edition, John Wiley & Sons, New York (1999). For example, a
hydroxy protected form is where at least one of the hydroxy groups
present in a compound is protected with a hydroxy protecting group.
Likewise, amines and other reactive groups may similarly be
protected.
"Solvate" refers to a compound (e.g., a compound selected from
Formula I or a pharmaceutically acceptable salt thereof) in
physical association with one or more molecules of a
pharmaceutically acceptable solvent. It will be understood that "a
compound of Formula I" encompass the compound of Formula I and
solvates of the compound, as well as mixtures thereof.
"Substituted" means that the referenced group may be substituted
with one or more additional group(s) individually and independently
selected from acyl, alkyl, alkylaryl, cycloalkyl, aralkyl, aryl,
carbohydrate, carbonate, heteroaryl, heterocycloalkyl, hydroxy,
alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo,
carbonyl, ester, thiocarbonyl, isocyanato, thiocyanato,
isothiocyanato, nitro, oxo, perhaloalkyl, perfluoroalkyl,
phosphate, silyl, sulfonyl, sulfonyl, sulfonamidyl, sulfoxyl,
sulfonate, urea, and amino, including mono- and di-substituted
amino groups, and the protected derivatives thereof. Di-substituted
amino groups encompass those which form a ring together with the
nitrogen of the amino group, such as for instance, morpholino. The
substituents themselves may be substituted, for example, a
cycloakyl substituent may have a halide substituted at one or more
ring carbons, and the like. The protecting groups that may form the
protective derivatives of the above substituents are known to those
of skill in the art and may be found in references such as Greene
and Wuts, above.
"Sulfanyl" refers to the groups: --S-(optionally substituted
alkyl), --S-(optionally substituted aryl), --S-(optionally
substituted heteroaryl), and --S-(optionally substituted
heterocycloalkyl).
"Sulfinyl" refers to the groups: --S(O)--H, --S(O)-(optionally
substituted alkyl), --S(O)-(optionally substituted amino),
--S(O)-(optionally substituted aryl), --S(O)-(optionally
substituted heteroaryl), and --S(O)-(optionally substituted
heterocycloalkyl).
"Sulfonyl" refers to the groups: --S(O.sub.2)--H,
--S(O.sub.2)-(optionally substituted alkyl),
--S(O.sub.2)-(optionally substituted amino),
--S(O.sub.2)-(optionally substituted aryl),
--S(O.sub.2)-(optionally substituted heteroaryl), and
--S(O.sub.2)-(optionally substituted heterocycloalkyl).
"Sulfonamidyl" or "sulfonamido" refers to a --S(.dbd.O).sub.2--NRR
radical, where each R is selected independently from the group
consisting of hydrogen, alkyl, cycloalkyl, aryl, heteroaryl (bonded
through a ring carbon) and heteroalicyclic (bonded through a ring
carbon). The R groups in --NRR of the --S(.dbd.O).sub.2--NRR
radical may be taken together with the nitrogen to which it is
attached to form a 4-, 5-, 6-, or 7-membered ring. In some
embodiments, it is a C.sub.1-C.sub.10 sulfonamido, wherein each R
in sulfonamido contains 1 carbon, 2 carbons, 3 carbons, or 4
carbons total. A sulfonamido group is optionally substituted by one
or more of the substituents described for alkyl, cycloalkyl, aryl,
heteroaryl respectively
"Sulfoxyl" refers to a --S(.dbd.O).sub.2OH radical.
"Sulfonate" refers to a --S(.dbd.O).sub.2--OR radical, where R is
selected from the group consisting of alkyl, cycloalkyl, aryl,
heteroaryl (bonded through a ring carbon) and heteroalicyclic
(bonded through a ring carbon). A sulfonate group is optionally
substituted on R by one or more of the substituents described for
alkyl, cycloalkyl, aryl, heteroaryl respectively.
Where substituent groups are specified by their conventional
chemical formulae, written from left to right, they equally
encompass the chemically identical substituents that would result
from writing the structure from right to left, e.g., --CH.sub.2O--
is equivalent to --OCH.sub.2--.
Compounds of the present invention also include crystalline and
amorphous forms of those compounds, including, for example,
polymorphs, pseudopolymorphs, solvates, hydrates, unsolvated
polymorphs (including anhydrates), conformational polymorphs, and
amorphous forms of the compounds, as well as mixtures thereof.
"Crystalline form," "polymorph," and "novel form" may be used
interchangeably herein, and are meant to include all crystalline
and amorphous forms of the compound, including, for example,
polymorphs, pseudopolymorphs, solvates, hydrates, unsolvated
polymorphs (including anhydrates), conformational polymorphs, and
amorphous forms, as well as mixtures thereof, unless a particular
crystalline or amorphous form is referred to.
Chemical entities include, but are not limited to, compounds of
Formula I, I-1, IV, IV-A, V, V-A, V-A2, V-B, VI or VI-A, and all
pharmaceutically acceptable forms thereof. Pharmaceutically
acceptable forms of the compounds recited herein include
pharmaceutically acceptable salts, chelates, non-covalent
complexes, prodrugs, and mixtures thereof. In certain embodiments,
the compounds described herein are in the form of pharmaceutically
acceptable salts. Hence, the terms "chemical entity" and "chemical
entities" also encompass pharmaceutically acceptable salts,
chelates, non-covalent complexes, prodrugs, and mixtures.
In addition, if the compound of Formula I is obtained as an acid
addition salt, the free base can be obtained by basifying a
solution of the acid salt. Conversely, if the product is a free
base, an addition salt, particularly a pharmaceutically acceptable
addition salt, may be produced by dissolving the free base in a
suitable organic solvent and treating the solution with an acid, in
accordance with conventional procedures for preparing acid addition
salts from base compounds. Those skilled in the art will recognize
various synthetic methodologies that may be used to prepare
non-toxic pharmaceutically acceptable addition salts.
In one aspect, the present invention provides a compound of Formula
I:
##STR00019##
or its pharmaceutically acceptable salt thereof, wherein
W.sub.d is heterocycloalkyl, aryl or heteroaryl;
B is alkyl, amino, heteroalkyl, or a moiety of Formula II;
##STR00020##
wherein W.sub.c is aryl, heteroaryl, heterocycloalkyl, or
cycloalkyl, and
q is an integer of 0, 1, 2, 3, or 4;
X is absent or is --(CH(R.sup.9)).sub.z and z is an integer of 1,
2, 3, or 4;
Y is absent, --O--, --S--, --S(.dbd.O)--, --S(.dbd.O).sub.2--,
--N(R.sup.9)--, --C(.dbd.O)--(CHR.sup.9).sub.z--, --C(.dbd.O)--,
--N(R.sup.9)--C(.dbd.O)--, or --N(R.sup.9)--C(.dbd.O)NH--,
--N(R.sup.9)C(R.sup.9).sub.2--, or
--C(.dbd.O)--(CHR.sup.9).sub.z--;
R.sup.1 is hydrogen, alkyl, heteroalkyl, alkenyl, alkynyl,
cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,
heteroarylalkyl, alkoxy, amido, amino, acyl, acyloxy,
alkoxycarbonyl, sulfonamido, halo, cyano, hydroxy, nitro,
phosphate, urea, or carbonate;
R.sup.2 is alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl,
heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl,
alkoxy, amido, amino, acyl, acyloxy, alkoxycarbonyl, sulfonamido,
halo, cyano, hydroxy, nitro, phosphate, urea, or carbonate;
R.sup.3 is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,
heterocycloalkyl, alkoxy, amido, amino, acyl, acyloxy,
alkoxycarbonyl, sulfonamido, halo, cyano, hydroxy, nitro, aryl, or
heteroaryl;
R.sup.5, R.sup.6, R.sup.7, and R.sup.8 are independently hydrogen,
C.sub.1-C.sub.4alkyl, C.sub.2-C.sub.5alkenyl,
C.sub.2-C.sub.5alkynyl, C.sub.3-C.sub.5cycloalkyl,
C.sub.1-C.sub.4heteroalkyl, C.sub.1-C.sub.4alkoxy,
C.sub.1-C.sub.4amido, amino, acyl, C.sub.1-C.sub.4acyloxy,
C.sub.1-C.sub.4sulfonamido, halo, cyano, hydroxy or nitro; and
each instance of R.sup.9 is independently hydrogen,
C.sub.1-C.sub.10alkyl, C.sub.3-C.sub.7cycloalkyl, heterocycloalkyl,
or C.sub.2-C.sub.10heteroalkyl.
In some embodiments, B is unsubstituted or substituted alkyl,
including but not limited to --(CH.sub.2).sub.2--NR.sup.aR.sup.a,
wherein each R.sup.a is independently hydrogen, alkyl, fluoroalkyl,
carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,
heterocycloalkylalkyl, heteroaryl or heteroarylalkyl, or
NR.sup.aR.sup.a are combined together to form a cyclic moiety,
which includes but is not limited to piperidinyl, piperazinyl, and
morpholinyl. In some embodiments, B is unsubstituted or substituted
amino. In some embodiments, B is unsubstituted or substituted
heteroalkyl.
##STR00021##
In some embodiments, B is a moiety of Formula II and wherein
W.sub.c is a member selected from the group consisting of
unsubstituted or substituted aryl, substituted phenyl,
unsubstituted or substituted heteroaryl including but not limited
to pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, pyrimidin-4-yl,
pyrimidin-2-yl, pyrimidin-5-yl, or pyrazin-2-yl, unsubstituted or
substituted monocyclic heteroaryl, unsubstituted or substituted
bicyclic heteroaryl, a heteroaryl comprising two heteroatoms as
ring atoms, unsubstituted or substituted heteroaryl comprising a
nitrogen ring atom, heteroaryl comprising two nitrogen ring atoms,
heteroaryl comprising a nitrogen and a sulfur as ring atoms,
unsubstituted or substituted heterocycloalkyl including but not
limited to morpholinyl, tetrahydropyranyl, piperazinyl, and
piperidinyl, unsubstituted or substituted cycloalkyl including but
not limited to cyclopentyl and cyclohexyl.
In some embodiments, B is one of the following moieties:
##STR00022## ##STR00023## ##STR00024## ##STR00025## ##STR00026##
##STR00027##
In some embodiments, B is substituted by one or more of alkyl,
heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl,
heteroaryl alkoxy, amido, amino, acyl, acyloxy, alkoxycarbonyl,
sulfonamido, halo, cyano, hydroxy or nitro, each of which alkyl,
heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl,
heteroaryl, alkoxy, amido, amino, acyl, acyloxy, or sulfonamido,
may itself be substituted.
In some embodiments, R.sup.1 is a member selected from the group
consisting of hydrogen, unsubstituted or substituted alkyl,
unsubstituted or substituted heteroalkyl, unsubstituted or
substituted alkenyl, unsubstituted or substituted alkynyl,
unsubstituted or substituted cycloalkyl, or unsubstituted or
substituted heterocycloalkyl. In some embodiments, R.sup.1 is
unsubstituted or substituted aryl, unsubstituted or substituted
arylalkyl, unsubstituted or substituted heteroaryl, or
unsubstituted or substituted heteroarylalkyl. In some embodiments,
R.sup.1 is unsubstituted or substituted alkoxy, unsubstituted or
substituted amido, unsubstituted or substituted amino. In some
embodiments, R.sup.1 is unsubstituted or substituted acyl,
unsubstituted or substituted acyloxy, unsubstituted or substituted
alkoxycarbonyl, or unsubstituted or substituted sulfonamido. In
some embodiments, R.sup.1 is halo which includes --Cl, --F, --I,
and --Br. In some embodiments, R.sup.1 is selected from the group
consisting of cyano, hydroxy, nitro, unsubstituted or substituted
phosphate, unsubstituted or substituted urea, and carbonate.
In some embodiments, when R.sup.1 is alkyl, R.sup.1 is methyl,
ethyl, propyl, isopropyl, n-butyl, tert-butyl, sec-butyl, pentyl,
hexyl or heptyl.
In some embodiments, when R.sup.1 is alkyl, heteroalkyl, alkenyl,
alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,
heteroarylalkyl, alkoxy, amido, amino, acyl, acyloxy,
alkoxycarbonyl, sulfonamido, or hydroxy, R.sup.1 is substituted by
phosphate, or unsubstituted urea, or substituted urea, or carbonic
acid, or carbonate.
In some embodiments, when R.sup.1 is alkyl, heteroalkyl, alkenyl,
alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,
heteroarylalkyl, alkoxy, amido, amino, acyl, acyloxy,
alkoxycarbonyl, or sulfonamido, R.sup.1 is substituted by one or
more of alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl,
heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl,
alkoxy, amido, amino, acyl, acyloxy, alkoxycarbonyl, sulfonamido,
halo, cyano, hydroxy or nitro, each of which alkyl, heteroalkyl,
alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl,
heteroaryl, heteroarylalkyl, alkoxy, amido, amino, acyl, acyloxy,
alkoxycarbonyl, or sulfonamido may itself be substituted.
In some embodiments, R.sup.2 is a member selected from the group
consisting of unsubstituted or substituted alkyl, unsubstituted or
substituted heteroalkyl, unsubstituted or substituted alkenyl,
unsubstituted or substituted alkynyl, unsubstituted or substituted
cycloalkyl, and unsubstituted or substituted heterocycloalkyl. In
some embodiments, R.sup.2 is unsubstituted or substituted aryl,
unsubstituted or substituted arylalkyl, unsubstituted or
substituted heteroaryl, or unsubstituted or substituted
heteroarylalkyl. In some embodiments, R.sup.2 is unsubstituted or
substituted alkoxy, unsubstituted or substituted amido,
unsubstituted or substituted amino. In some embodiments, R.sup.2 is
unsubstituted or substituted acyl, unsubstituted or substituted
acyloxy, unsubstituted or substituted alkoxycarbonyl, or
unsubstituted or substituted sulfonamido. In some embodiments,
R.sup.2 is halo, which is --I, --F, --Cl, or --Br. In some
embodiments, R.sup.2 is selected from the group consisting of
cyano, hydroxy, nitro, a carbonic acid, and a carbonate. In some
embodiments, R.sup.2 is unsubstituted or substituted phosphate. In
some embodiments, R.sup.2 is unsubstituted or substituted urea. In
some embodiments, when R.sup.2 is alkyl, R.sup.2 is methyl, ethyl,
propyl, isopropyl, n-butyl, tert-butyl, sec-butyl, pentyl, hexyl or
heptyl.
In some embodiments, when R.sup.2 is alkyl, heteroalkyl, alkenyl,
alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,
heteroarylalkyl, alkoxy, amido, amino, acyl, acyloxy,
alkoxycarbonyl, sulfonamido, or hydroxy, it is substituted by
phosphate, substituted by urea, or substituted by carbonate.
In some embodiments, when R.sup.2 is alkyl, heteroalkyl, alkenyl,
alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,
heteroarylalkyl, alkoxy, amido, amino, acyl, acyloxy,
alkoxycarbonyl, or sulfonamido, it is substituted by one or more of
alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,
aryl, heteroaryl, alkoxy, amido, amino, acyl, acyloxy,
alkoxycarbonyl, sulfonamido, halo, cyano, hydroxy or nitro, each of
which alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl,
heterocycloalkyl, aryl, heteroaryl, alkoxy, amido, amino, acyl,
acyloxy, alkoxycarbonyl, or sulfonamido may itself be
substituted.
In some embodiments, q is an integer of 0. In some embodiments, q
is an integer of 1. In some embodiments, q is an integer of 2. In
some embodiments, q is an integer of 3. In some embodiments, q is
an integer of 4.
In some embodiments of the compound of Formula I, R.sup.3 is a
member selected from the group consisting of hydrogen,
unsubstituted or substituted alkyl, unsubstituted or substituted
alkenyl, and unsubstituted or substituted alkynyl. In some
embodiments, R.sup.3 is unsubstituted or substituted aryl,
unsubstituted or substituted heteroaryl, unsubstituted or
substituted cycloalkyl, or unsubstituted or substituted
heterocycloalkyl. In some embodiments, R.sup.3 is unsubstituted or
substituted alkoxy, unsubstituted or substituted amido,
unsubstituted or substituted amino. In some embodiments, R.sup.3 is
unsubstituted or substituted acyl, unsubstituted or substituted
acyloxy, unsubstituted or substituted alkoxycarbonyl, or
unsubstituted or substituted sulfonamido. In some embodiments,
R.sup.3 is halo, which is --I, --F, --Cl, or --Br.
In some embodiments, R.sup.3 is selected from the group consisting
of cyano, hydroxy, and nitro. In some embodiments, when R.sup.3 is
alkyl, R.sup.3 is methyl, ethyl, propyl, isopropyl, n-butyl,
tert-butyl, sec-butyl, pentyl, hexyl or heptyl. In some
embodiments, R.sup.3 is --CF.sub.3.
In some embodiments, when R.sup.3 is alkyl, alkenyl, alkynyl, aryl,
heteroaryl, cycloalkyl, heterocycloalkyl, alkoxy, amido, amino,
acyl, acyloxy, alkoxycarbonyl, or sulfonamido, it is substituted
with one or more of alkyl, heteroalkyl, alkenyl, alkynyl,
cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, amido,
amino, acyl, acyloxy, alkoxycarbonyl, sulfonamido, halo, cyano,
hydroxy or nitro, each of which alkyl, heteroalkyl, alkenyl,
alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy,
amido, amino, acyl, acyloxy, alkoxycarbonyl, or sulfonamido may
itself be substituted.
In some embodiments of the compound of Formula I, R.sup.5 is
hydrogen, unsubstituted or substituted alkyl (including but not
limited to unsubstituted or substituted C.sub.1-C.sub.4alkyl). In
some embodiments, R.sup.5 is unsubstituted or substituted alkenyl
including but not limited to unsubstituted or substituted
C.sub.2-C.sub.5alkenyl. In some embodiments, R.sup.5 is
unsubstituted or substituted alkynyl including but not limited to
unsubstituted or substituted C.sub.2-C.sub.5alkynyl. In some
embodiments, R.sup.5 is unsubstituted or substituted cycloalkyl
including but not limited to unsubstituted or substituted
C.sub.3-C.sub.5cycloalkyl. In some embodiments, R.sup.5 is
unsubstituted or substituted heterocycloalkyl. In some embodiments,
R.sup.5 is unsubstituted or substituted heteroalkyl including but
not limited to unsubstituted or substituted
C.sub.1-C.sub.4heteroalkyl. In some embodiments, R.sup.5 is
unsubstituted or substituted alkoxy including but not limited to
unsubstituted or substituted C.sub.1-C.sub.4alkoxy. In some
embodiments, R.sup.5 is unsubstituted or substituted amido
including but not limited to unsubstituted or substituted
C.sub.1-C.sub.4amido. In some embodiments, R.sup.5 is unsubstituted
or substituted amino. In some embodiments, R.sup.5 is unsubstituted
or substituted acyl, unsubstituted or substituted acyloxy,
unsubstituted or substituted C.sub.1-C.sub.4acyloxy, unsubstituted
or substituted alkoxycarbonyl, unsubstituted or substituted
sulfonamido, or unsubstituted or substituted
C.sub.1-C.sub.4sulfonamido. In some embodiments, R.sup.5 is halo,
which is --I, --F, --Cl, or --Br. In some embodiments, R.sup.5 is
selected from the group consisting of cyano, hydroxy, and nitro. In
some other embodiments, R.sup.5 is --CH.sub.3, --CH.sub.2CH.sub.3,
n-propyl, isopropyl, --OCH.sub.3, --OCH.sub.2CH.sub.3, or
--CF.sub.3.
In some embodiments, when R.sup.5 is alkyl, alkenyl, alkynyl,
cycloalkyl, heteroalkyl, acyl, alkoxy, amido, amino, acyloxy,
alkoxycarbonyl, or sulfonamido, R.sup.5 is optionally substituted
with one or more of alkyl, heteroalkyl, alkenyl, alkynyl,
cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, amido,
amino, acyl, acyloxy, alkoxycarbonyl, sulfonamido, halo, cyano,
hydroxy or nitro, each of which alkyl, heteroalkyl, alkenyl,
alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy,
amido, amino, acyl, acyloxy, alkoxycarbonyl, or sulfonamido may
itself be substituted.
In some embodiments of the compound of Formula I, R.sup.6 is
hydrogen, unsubstituted or substituted alkyl (including but not
limited to unsubstituted or substituted C.sub.1-C.sub.4alkyl). In
some embodiments, R.sup.6 is unsubstituted or substituted alkenyl
including but not limited to unsubstituted or substituted
C.sub.2-C.sub.5alkenyl. In some embodiments, R.sup.6 is
unsubstituted or substituted alkynyl including but not limited to
unsubstituted or substituted C.sub.2-C.sub.5alkynyl. In some
embodiments, R.sup.6 is unsubstituted or substituted cycloalkyl
including but not limited to unsubstituted or substituted
C.sub.3-C.sub.5cycloalkyl. In some embodiments, R.sup.6 is
unsubstituted or substituted heterocycloalkyl. In some embodiments,
R.sup.6 is unsubstituted or substituted heteroalkyl including but
not limited to unsubstituted or substituted
C.sub.1-C.sub.4heteroalkyl. In some embodiments, R.sup.6 is
unsubstituted or substituted alkoxy including but not limited to
unsubstituted or substituted C.sub.1-C.sub.4alkoxy. In some
embodiments, R.sup.6 is unsubstituted or substituted amido
including but not limited to unsubstituted or substituted
C.sub.1-C.sub.4amido. In some embodiments, R.sup.6 is unsubstituted
or substituted amino. In some embodiments, R.sup.6 is unsubstituted
or substituted acyl, unsubstituted or substituted acyloxy,
unsubstituted or substituted C.sub.1-C.sub.4acyloxy, unsubstituted
or substituted alkoxycarbonyl, unsubstituted or substituted
sulfonamido, or unsubstituted or substituted
C.sub.1-C.sub.4sulfonamido. In some embodiments, R.sup.6 is halo,
which is --I, --F, --Cl, or --Br. In some embodiments, R.sup.6 is
selected from the group consisting of cyano, hydroxy, and nitro. In
some other embodiments, R.sup.6 is --CH.sub.3, --CH.sub.2CH.sub.3,
n-propyl, isopropyl, --OCH.sub.3, --OCH.sub.2CH.sub.3, or
--CF.sub.3.
In some embodiments, when R.sup.6 is alkyl, alkenyl, alkynyl,
cycloalkyl, heteroalkyl, acyl, alkoxy, amido, amino, acyloxy,
alkoxycarbonyl, or sulfonamido, R.sup.6 is optionally substituted
with one or more of alkyl, heteroalkyl, alkenyl, alkynyl,
cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, amido,
amino, acyl, acyloxy, alkoxycarbonyl, sulfonamido, halo, cyano,
hydroxy or nitro, each of which alkyl, heteroalkyl, alkenyl,
alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy,
amido, amino, acyl, acyloxy, alkoxycarbonyl, or sulfonamido may
itself be substituted.
In some embodiments of the compound of Formula I, R.sup.7 is
hydrogen, unsubstituted or substituted alkyl (including but not
limited to unsubstituted or substituted C.sub.1-C.sub.4alkyl). In
some embodiments, R.sup.7 is unsubstituted or substituted alkenyl
including but not limited to unsubstituted or substituted
C.sub.2-C.sub.5alkenyl. In some embodiments, R.sup.7 is
unsubstituted or substituted alkynyl including but not limited to
unsubstituted or substituted C.sub.2-C.sub.5alkynyl. In some
embodiments, R.sup.7 is unsubstituted or substituted cycloalkyl
including but not limited to unsubstituted or substituted
C.sub.3-C.sub.5cycloalkyl. In some embodiments, R.sup.7 is
unsubstituted or substituted heterocycloalkyl. In some embodiments,
R.sup.7 is unsubstituted or substituted heteroalkyl including but
not limited to unsubstituted or substituted
C.sub.1-C.sub.4heteroalkyl. In some embodiments, R.sup.7 is
unsubstituted or substituted alkoxy including but not limited to
unsubstituted or substituted C.sub.1-C.sub.4alkoxy. In some
embodiments, R.sup.7 is unsubstituted or substituted amido
including but not limited to unsubstituted or substituted
C.sub.1-C.sub.4amido. In some embodiments, R.sup.7 is unsubstituted
or substituted amino. In some embodiments, R.sup.7 is unsubstituted
or substituted acyl, unsubstituted or substituted acyloxy,
unsubstituted or substituted C.sub.1-C.sub.4acyloxy, unsubstituted
or substituted alkoxycarbonyl, unsubstituted or substituted
sulfonamido, or unsubstituted or substituted
C.sub.1-C.sub.4sulfonamido. In some embodiments, R.sup.7 is halo,
which is --I, --F, --Cl, or --Br. In some embodiments, R.sup.7 is
selected from the group consisting of cyano, hydroxy, and nitro. In
some other embodiments, R.sup.7 is --CH.sub.3, --CH.sub.2CH.sub.3,
n-propyl, isopropyl, --OCH.sub.3, --OCH.sub.2CH.sub.3, or
--CF.sub.3.
In some embodiments, when R.sup.7 is alkyl, alkenyl, alkynyl,
cycloalkyl, heteroalkyl, acyl, alkoxy, amido, amino, acyloxy,
alkoxycarbonyl, or sulfonamido, R.sup.7 is optionally substituted
with one or more of alkyl, heteroalkyl, alkenyl, alkynyl,
cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, amido,
amino, acyl, acyloxy, alkoxycarbonyl, sulfonamido, halo, cyano,
hydroxy or nitro, each of which alkyl, heteroalkyl, alkenyl,
alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy,
amido, amino, acyl, acyloxy, alkoxycarbonyl, or sulfonamido may
itself be substituted.
In some embodiments of the compound of Formula I, R.sup.8 is
hydrogen, unsubstituted or substituted alkyl (including but not
limited to unsubstituted or substituted C.sub.1-C.sub.4alkyl). In
some embodiments, R.sup.8 is unsubstituted or substituted alkenyl
including but not limited to unsubstituted or substituted
C.sub.2-C.sub.5alkenyl. In some embodiments, R.sup.8 is
unsubstituted or substituted alkynyl including but not limited to
unsubstituted or substituted C.sub.2-C.sub.5alkynyl. In some
embodiments, R.sup.8 is unsubstituted or substituted cycloalkyl
including but not limited to unsubstituted or substituted
C.sub.3-C.sub.5cycloalkyl. In some embodiments, R.sup.8 is
unsubstituted or substituted heterocycloalkyl. In some embodiments,
R.sup.8 is unsubstituted or substituted heteroalkyl including but
not limited to unsubstituted or substituted
C.sub.1-C.sub.4heteroalkyl. In some embodiments, R.sup.8 is
unsubstituted or substituted alkoxy including but not limited to
unsubstituted or substituted C.sub.1-C.sub.4alkoxy. In some
embodiments, R.sup.8 is unsubstituted or substituted amido
including but not limited to unsubstituted or substituted
C.sub.1-C.sub.4amido. In some embodiments, R.sup.8 is unsubstituted
or substituted amino. In some embodiments, R.sup.8 is unsubstituted
or substituted acyl, unsubstituted or substituted acyloxy,
unsubstituted or substituted C.sub.1-C.sub.4acyloxy, unsubstituted
or substituted alkoxycarbonyl, unsubstituted or substituted
sulfonamido, or unsubstituted or substituted
C.sub.1-C.sub.4sulfonamido. In some embodiments, R.sup.8 is halo,
which is --I, --F, --Cl, or --Br. In some embodiments, R.sup.8 is
selected from the group consisting of cyano, hydroxy, and nitro. In
some other embodiments, R.sup.8 is --CH.sub.3, --CH.sub.2CH.sub.3,
n-propyl, isopropyl, --OCH.sub.3, --OCH.sub.2CH.sub.3, or
--CF.sub.3.
In some embodiments, when R.sup.8 is alkyl, alkenyl, alkynyl,
cycloalkyl, heteroalkyl, acyl, alkoxy, amido, amino, acyloxy,
alkoxycarbonyl, or sulfonamido, R.sup.8 is optionally substituted
with one or more of alkyl, heteroalkyl, alkenyl, alkynyl,
cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, amido,
amino, acyl, acyloxy, alkoxycarbonyl, sulfonamido, halo, cyano,
hydroxy or nitro, each of which alkyl, heteroalkyl, alkenyl,
alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy,
amido, amino, acyl, acyloxy, alkoxycarbonyl, or sulfonamido may
itself be substituted.
In some embodiments of the compound of Formula I, R.sup.5, R.sup.6,
R.sup.7, and R.sup.8 are H and the compound has a structure of
Formula I-1:
##STR00028##
In some embodiments of the compound of Formula I, X is absent. In
some embodiments, X is --(CH(R.sup.9)).sub.z, and z is an integer
of 1, 2, 3 or 4.
In some embodiments, R.sup.9 is unsubstituted or substituted alkyl
including but not limited to unsubstituted or substituted
C.sub.1-C.sub.10alkyl. In some embodiments, R.sup.9 is
unsubstituted or substituted cycloalkyl including but not limited
to unsubstituted or substituted C.sub.3-C.sub.7cycloalkyl. In some
embodiments, R.sup.9 is ethyl, methyl or hydrogen. In some
embodiments, R.sup.9 is unsubstituted or substituted
heterocycloalkyl including but not limited to unsubstituted or
substituted C.sub.2-C.sub.10heteroalkyl. In some embodiments,
R.sup.9 is unsubstituted or substituted heteroalkyl including but
not limited to unsubstituted or substituted
C.sub.2-C.sub.10heteroalkyl.
The invention also provides a compound of Formula I wherein R.sup.9
is hydrogen, and X is --CH.sub.2--, --CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2--, --CH(CH.sub.3)--, or
--CH(CH.sub.2CH.sub.3)--. In other embodiments, X is
--(CH(R.sup.9)).sub.z, R.sup.9 is not hydrogen, and z is an integer
of 1. When X is --CH(R.sup.9)-- and R.sup.9 is not hydrogen, then
the compound can adopt either an (S)- or (R)-stereochemical
configuration with respect to carbon X. In some embodiments, the
compound is a racemic mixture of (S)- and (R) isomers with respect
to carbon X. In other embodiments, the present invention provides a
mixture of compounds of Formula I wherein individual compounds of
the mixture exist predominately in an (S)- or (R)-isomeric
configuration. For example, the compound mixture has an
(S)-enantiomeric purity of greater than about 55%, about 60%, about
65%, about 70%, about 75%, about 80%, about 85%, about 90%, about
95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or
more at the X carbon. In other embodiments, the compound mixture
has an (S)-enantiomeric purity of greater than about 55% to about
99.5%, greater than about 60% to about 99.5%, greater than about
65% to about 99.5%, greater than about 70% to about 99.5%, greater
than about 75% to about 99.5%, greater than about 80% to about
99.5%, greater than about 85% to about 99.5%, greater than about
90% to about 99.5%, greater than about 95% to about 99.5%, greater
than about 96% to about 99.5%, greater than about 97% to about
99.5%, greater than about 98% to greater than about 99.5%, greater
than about 99% to about 99.5%, or more.
In other embodiments, the compound mixture has an (R)-enantiomeric
purity of greater than about 55%, about 60%, about 65%, about 70%,
about 75%, about 80%, about 85%, about 90%, about 95%, about 96%,
about 97%, about 98%, about 99%, about 99.5%, or more at the X
carbon. In some other embodiments, the compound mixture has an
(R)-enantiomeric purity of greater than about 55% to about 99.5%,
greater than about 60% to about 99.5%, greater than about 65% to
about 99.5%, greater than about 70% to about 99.5%, greater than
about 75% to about 99.5%, greater than about 80% to about 99.5%,
greater than about 85% to about 99.5%, greater than about 90% to
about 99.5%, greater than about 95% to about 99.5%, greater than
about 96% to about 99.5%, greater than about 97% to about 99.5%,
greater than about 98% to greater than about 99.5%, greater than
about 99% to about 99.5%, or more.
In other embodiments, the compound mixture contains identical
chemical entities except for their stereochemical orientations,
namely (S)- or (R)-isomers. For instance, in the compounds of
Formula I, when X is --CH(R.sup.9)--, and R.sup.9 is not hydrogen,
then the --CH(R.sup.9)-- is in an (S)- or (R)-stereochemical
orientation for each of the identical chemical entities. In some
embodiments, the mixture of identical chemical entities of Formula
I is a racemic mixture of (S)- and (R)-isomers at the carbon
represented by X. In another embodiment, the mixture of the
identical chemical entities (except for their stereochemical
orientations), contain predominately (S)-isomers or predominately
(R)-isomers. For example, the (S)-isomers in the mixture of
identical chemical entities are present at about 55%, about 60%,
about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,
about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%,
or more, relative to the (R)-isomers. In some embodiments, the
(S)-isomers in the mixture of identical chemical entities are
present at an (S)-enantiomeric purity of greater than about 55% to
about 99.5%, greater than about 60% to about 99.5%, greater than
about 65% to about 99.5%, greater than about 70% to about 99.5%,
greater than about 75% to about 99.5%, greater than about 80% to
about 99.5%, greater than about 85% to about 99.5%, greater than
about 90% to about 99.5%, greater than about 95% to about 99.5%,
greater than about 96% to about 99.5%, greater than about 97% to
about 99.5%, greater than about 98% to greater than about 99.5%,
greater than about 99% to about 99.5%, or more.
In another embodiment, the (R)-isomers in the mixture of identical
chemical entities (except for their stereochemical orientations),
are present at about 55%, about 60%, about 65%, about 70%, about
75%, about 80%, about 85%, about 90%, about 95%, about 96%, about
97%, about 98%, about 99%, about 99.5%, or more, relative to the
(S)-isomers. In some embodiments, the (R)-isomers in the mixture of
identical chemical entities (except for their stereochemical
orientations), are present at a (R)-enantiomeric purity greater
than about 55% to about 99.5%, greater than about 60% to about
99.5%, greater than about 65% to about 99.5%, greater than about
70% to about 99.5%, greater than about 75% to about 99.5%, greater
than about 80% to about 99.5%, greater than about 85% to about
99.5%, greater than about 90% to about 99.5%, greater than about
95% to about 99.5%, greater than about 96% to about 99.5%, greater
than about 97% to about 99.5%, greater than about 98% to greater
than about 99.5%, greater than about 99% to about 99.5%, or
more.
In some embodiments, the compound of Formula I, X is
--CH(R.sup.9)--, R.sup.9 is methyl or ethyl, and the compound is
the (S)-isomer.
In some embodiments of the compound of Formula I, Y is absent. In
some embodiments, Y is --O--, --S--, --S(.dbd.O)--,
--S(.dbd.O).sub.2--, --C(.dbd.O)--, --N(R.sup.9)(C.dbd.O)--,
--N(R.sup.9)(C.dbd.O)NH--, --N(R.sup.9)C(R.sup.9).sub.2-- (such as
--N(R.sup.9)CH.sub.2--, specifically --N(CH.sub.3)CH.sub.2--,
N(CH(CH.sub.3).sub.2)CH.sub.2-- or N(CH.sub.2CH.sub.3)CH.sub.2--),
--N(R.sup.9)--, --N(CH.sub.3)--, --N(CH.sub.2CH.sub.3)--, or
--N(CH(CH.sub.3).sub.2)--. In some embodiments, Y is
--C(.dbd.O)--(CHR.sup.9).sub.z-- and z is an integer of 1, 2, 3, or
4.
In some embodiments, at least one of X and Y is present. In some
embodiments of the compound of Formula I, --XY-- is --CH.sub.2--,
--CH.sub.2--N(CH.sub.3), --CH.sub.2--N(CH.sub.2CH.sub.3),
--CH(CH.sub.3)--NH--, (S)--CH(CH.sub.3)--NH--, or
(R)--CH(CH.sub.3)--NH--. In other embodiments, X--Y is
--N(CH.sub.3)_CH.sub.2--, N(CH.sub.2CH.sub.3) CH.sub.2--,
--N(CH(CH.sub.3).sub.2)CH.sub.2--, or --NHCH.sub.2--. The invention
provides other compounds of Formula I wherein when X-Y is X is
--(CH(R.sup.9)).sub.zN(R.sup.9)--, z is an integer of 1, 2, 3 or 4,
and --N(R.sup.9)-- is not --NH--, then --XY-- is not connected to
purinyl.
In some embodiments, W.sub.d in a formula disclosed herein
(including but not limited to I, I-1, IV, IV-A, V, V-A, V-A2, V-B,
VI and VI-A), is a member selected from the group consisting of
unsubstituted or substituted heterocycloalkyl, unsubstituted or
substituted aryl, and unsubstituted or substituted heteroaryl.
In various embodiments, W.sub.d is unsubstituted or substituted
monocyclic heteroaryl (including but not limited to pyrimidinyl,
pyrrolyl, pyrazinyl, triazinyl, or pyridazinyl) or unsubstituted or
substituted bicyclic heteroaryl.
In some embodiments, W.sub.d is a monocyclic heteroaryl of the
following formula:
##STR00029## wherein R.sub.a' is hydrogen, halo, phosphate, urea, a
carbonate, unsubstituted or substituted amino, unsubstituted or
substituted alkyl, unsubstituted or substituted alkenyl,
unsubstituted or substituted alkynyl, unsubstituted or substituted
cycloalkyl, unsubstituted or substituted heteroalkyl, or
unsubstituted or substituted heterocycloalkyl; and R.sup.12 is H,
unsubstituted or substituted alkyl, unsubstituted or substituted
cyano, unsubstituted or substituted alkynyl, unsubstituted or
substituted alkenyl, halo, unsubstituted or substituted aryl,
unsubstituted or substituted heteroaryl, unsubstituted or
substituted heterocycloalkyl, unsubstituted or substituted
cycloalkyl, unsubstituted or substituted amino, carboxylic acid,
unsubstituted or substituted alkoxycarbonyl, unsubstituted or
substituted amido, unsubstituted or substituted acyl, or
unsubstituted or substituted sulfonamido. The invention provides
monocyclic heteroaryl W.sub.d including but not limited to one of
the following formulae:
##STR00030##
In some embodiments, W.sub.d in a formula disclosed herein
(including but not limited to I, I-1, IV, IV-A, V, V-A, V-A2, V-B,
VI and VI-A), is a bicyclic heteroaryl having at least one
heteroatom, e.g., a bicyclic heteroaryl having at least one
nitrogen ring atom. In some embodiments, W.sub.d is a bicyclic
heteroaryl having at least two heteroatoms, e.g., a bicyclic
heteroaryl having at least two nitrogen ring atoms. In some
embodiments, W.sub.d is a bicyclic heteroaryl having two
heteroatoms in the ring which is connected to XY. In some
embodiments, W.sub.d is a bicyclic heteroaryl having two nitrogen
ring atoms in the ring to which XY is connected. In some
embodiments, W.sub.d is a bicyclic heteroaryl having four
heteroatoms, e.g, a bicyclic heteroaryl having four nitrogen ring
atoms. In some embodiments, W.sub.d is unsubstituted or substituted
4-amino-1H-pyrazolo[3,4-d]pyrimidin-1-yl, unsubstituted or
substituted 7-amino-2-methyl-2H-pyrazolo[4,3-d]pyrimidin-3-yl.
unsubstituted or substituted 6-methylenyl-9H-purin-6-yl, or
unsubstituted or substituted 6-amino-9H-purin-9-yl.
In some embodiments W.sub.d is one of the following:
##STR00031## ##STR00032## ##STR00033## wherein R.sup.a' is
hydrogen, halo, phosphate, urea, a carbonate, unsubstituted or
substituted amino, unsubstituted or substituted alkyl,
unsubstituted or substituted alkenyl, unsubstituted or substituted
alkynyl, unsubstituted or substituted cycloalkyl, unsubstituted or
substituted heteroalkyl, or unsubstituted or substituted
heterocycloalkyl; R.sup.11 is hydrogen, unsubstituted or
substituted alkyl, halo (which includes --I, --F, --Cl, or --Br),
unsubstituted or substituted amino, unsubstituted or substituted
amido, hydroxy, or unsubstituted or substituted alkoxy, phosphate,
unsubstituted or substituted urea, or carbonate; and R.sup.12 is H,
unsubstituted or substituted alkyl, unsubstituted or substituted
cyano, unsubstituted or substituted alkynyl, unsubstituted or
substituted alkenyl, halo, unsubstituted or substituted aryl,
unsubstituted or substituted heteroaryl, unsubstituted or
substituted heterocycloalkyl, unsubstituted or substituted
cycloalkyl, unsubstituted or substituted amino, carboxylic acid,
unsubstituted or substituted alkoxycarbonyl, unsubstituted or
substituted amido, unsubstituted or substituted acyl, or
unsubstituted or substituted sulfonamido.
In some embodiments of W.sub.d of the compounds of Formula I, when
R.sup.a' is alkyl, alkynyl, cycloalkyl, heteroalkyl, or
heterocycloalkyl, it is substituted by phosphate, urea, or
carbonate.
In some embodiments of W.sub.d of the compounds of Formula I, when
R.sup.11 is alkyl, amino, amido, hydroxy, or alkoxy, it is
substituted by phosphate, urea, or carbonate.
In some embodiments of the compound of Formula I, --X--Y--W.sub.d
is one of the following moieties:
##STR00034## ##STR00035## ##STR00036## ##STR00037## ##STR00038##
##STR00039## ##STR00040## ##STR00041## ##STR00042## ##STR00043##
##STR00044## ##STR00045## ##STR00046## ##STR00047## ##STR00048##
##STR00049## ##STR00050## ##STR00051##
In some embodiments of the compound of Formula I, R.sup.12 is a
member of the group consisting of hydrogen, cyano, halo,
unsubstituted or substituted alkyl, unsubstituted or substituted
alkynyl, and unsubstituted or substituted alkenyl. In some
embodiments, R.sup.12 is unsubstituted or substituted aryl. In some
embodiments, R.sup.12 is unsubstituted or substituted heteroaryl,
which includes but is not limited to heteroaryl having a 5 membered
ring, heteroaryl having a six membered ring, heteroaryl with at
least one nitrogen ring atom, heteroaryl with two nitrogen ring
atoms, monocylic heteroaryl, and bicylic heteroaryl. In some
embodiments, R.sup.12 is unsubstituted or substituted
heterocycloalkyl, which includes but is not limited to
heterocycloalkyl with one nitrogen ring atom, heterocycloalkyl with
one oxygen ring atom, R.sup.12 is heterocycloalkyl with one sulfur
ring atom, 5 membered heterocycloalkyl, 6 membered
heterocycloalkyl, saturated heterocycloalkyl, unsaturated
heterocycloalkyl, heterocycloalkyl having an unsaturated moiety
connected to the heterocycloalkyl ring, heterocycloalkyl
substituted by oxo, and heterocycloalkyl substituted by two oxo. In
some embodiments, R.sup.12 is unsubstituted or substituted
cycloalkyl, including but not limited to cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, cycloalkyl substituted by one oxo,
cycloalkyl having an unsaturated moiety connected to the cycloalkyl
ring. In some embodiments, R.sup.12 is unsubstituted or substituted
amido, carboxylic acid, unsubstituted or substituted acyloxy,
unsubstituted or substituted alkoxycarbonyl, unsubstituted or
substituted acyl, or unsubstituted or substituted sulfonamido.
In some embodiments, when R.sup.12 is alkyl, alkynyl, alkenyl,
aryl, heteroaryl, heterocycloalkyl, or cycloalkyl, it is
substituted with phosphate. In some embodiments, when R.sup.12 is
alkyl, alkynyl, alkenyl, aryl, heteroaryl, heterocycloalkyl, or
cycloalkyl, it is substituted with urea. In some embodiments, when
R.sup.12 is alkyl, alkynyl, alkenyl, aryl, heteroaryl,
heterocycloalkyl, or cycloalkyl, it is substituted with
carbonate.
In some embodiments, when R.sup.12 is alkyl, alkynyl, alkenyl,
aryl, heteroaryl, heterocycloalkyl, cycloalkyl, alkoxycarbonyl,
amido, acyloxy, acyl, or sulfonamido, it is substituted with one or
more of alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl,
heterocycloalkyl, aryl, heteroaryl, alkoxy, amido, amino, acyl,
acyloxy, alkoxycarbonyl, sulfonamido, halo, cyano, hydroxy or
nitro, each of which alkyl, heteroalkyl, alkenyl, alkynyl,
cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, amido,
amino, acyl, acyloxy, aloxycarbonyl, or sulfonamido may itself be
substituted.
In some embodiments of the compound of Formula I, R.sup.12 of
W.sub.d is one of the following moieties:
##STR00052## ##STR00053## ##STR00054## ##STR00055##
In some embodiments of the compound of Formula I, W.sub.d is a
pyrazolopyrimidine of Formula III:
##STR00056## wherein R.sup.11 is H, alkyl, halo, amino, amido,
hydroxy, or alkoxy, and R.sup.12 is H, alkyl, alkynyl, alkenyl,
halo, aryl, heteroaryl, heterocycloalkyl, or cycloalkyl. In some
embodiments, R.sup.11 is amino and R.sup.12 is H, alkyl, alkynyl,
alkenyl, halo, aryl, heteroaryl, heterocycloalkyl, or cycloalkyl.
In some embodiments, R.sup.11 is amino and R.sup.12 is alkyl, halo,
aryl, heteroaryl, heterocycloalkyl, or cycloalkyl. In some
embodiments, R.sup.11 is amino and R.sup.12 is monocyclic
heteroaryl. In some embodiments, R.sup.11 is amino and R.sup.12 is
bicyclic heteroaryl. In some embodiments, R.sup.11 is amino and
R.sup.12 is cyano, amino, carboxylic acid, acyloxy, alkoxycarbonyl,
or amido.
In some embodiments of the invention, the compound of Formula I is
a compound having a structure of Formula IV:
##STR00057## In some embodiments of the compound of Formula IV,
R.sup.11 is H, alkyl, halo, amino, amido, hydroxy, or alkoxy, and
R.sup.12 is H, alkyl, alkynyl, alkenyl, halo, aryl, heteroaryl,
heterocycloalkyl, or cycloalkyl. In another embodiment, R.sup.11 is
amino and R.sup.12 is alkyl, alkenyl, heteroaryl, aryl, or
heterocycloalkyl. In some embodiments, R.sup.11 is amino and
R.sup.12 is cyano, amino, carboxylic acid, alkoxycarbonyl, or
amido. In some embodiments, the compound of Formula IV is a
compound of Formula IV-A:
##STR00058##
The invention also provides compounds of Formula I having a
structure of any of Formulae V, V-A1, V-A2, V-B, VI, VI-A, VII-A1,
VII-A2, VIII-A1, VIII-A2, IX-A1, IX-A2, X-A1, X-A2, XI-A1, XI-A2,
XII-A, XII-A1, XII-A2, XIII-A, XIII-A1, XIII-A2, XIV-A, XIV-A1,
XIV-A2, XV-A, XV-A1, XV-A2, XVI-A, XVI-A1, XVI-A2, XVII-A, XVII-A1,
XVII-A2, XVIII-A, XVIII-A1, or XVIII-A2:
##STR00059## ##STR00060## ##STR00061## ##STR00062## ##STR00063##
##STR00064## ##STR00065## ##STR00066## ##STR00067##
##STR00068##
Any of the disclosed elements and their substituents for the
compounds of Formula I can be used in any combination.
In one aspect, for the compounds of Formula I, R.sub.3 is H,
CH.sub.3, CF.sub.3, Cl, or F; and B is a moiety of Formula II:
##STR00069## wherein W.sub.c is aryl, heteroaryl, heterocycloalkyl,
or cycloalkyl; R.sup.1 is H, --F, --Cl, --CN, --CH.sub.3,
isopropyl, --CF.sub.3, --OCH.sub.3, nitro, or phosphate; R.sup.2 is
halo, hydroxy, cyano, or nitro; q is an integer of 0, 1, 2, 3, or
4; R.sup.5, R.sup.6, R.sup.7, and R.sup.8 are H; X is absent or
(CH.sub.2).sub.z; z is 1; Y is absent or --N(R.sup.9)--; R.sup.9 is
hydrogen, C.sub.1-C.sub.10alkyl, C.sub.3-C.sub.7cycloalkyl, or
C.sub.2-C.sub.10heteroalkyl; at least one of X and Y is present;
and W.sub.d is pyrazolopyrimidine or purine. In some embodiments,
when X and Y are present and W.sub.d is purine, then --N(R.sup.9)--
is --NH--.
In another aspect, for the compounds of Formula I, R.sub.3 is H,
CH.sub.3, CF.sub.3, Cl, or F; B is a moiety of Formula II which is
aryl, heteroaryl, heterocycloalkyl, or cycloalkyl, R.sup.1 is H,
--F, --Cl, --CN, --CH.sub.3, isopropyl, --CF.sub.3, --OCH.sub.3,
nitro, or phosphate; R.sup.2 is halo, hydroxy, cyano, or nitro; q
is 0, 1 or 2; R.sup.5, R.sup.6, R.sup.7, and R.sup.8 are H; X is
absent or (CH.sub.2).sub.z; z is 1; Y is absent or --N(R.sup.9)--;
R.sup.9 is hydrogen, methyl, or ethyl; at least one of X and Y is
present; W.sub.d is:
##STR00070## R.sup.11 is amino; and R.sup.12 is H, alkyl, alkynyl,
alkenyl, halo, aryl, heteroaryl, heterocycloalkyl, or cycloalkyl.
In some embodiments, when X and Y are present and W.sub.d is
purine, then --N(R.sup.9)-- is --NH--.
In another aspect, for the compounds of Formula I, R.sub.3 is H,
CH.sub.3, CF.sub.3, Cl, or F; B is a moiety of Formula II, which is
aryl, heteroaryl, heterocycloalkyl, or cycloalkyl, R.sup.1 is H,
--F, --Cl, --CN, --CH.sub.3, isopropyl, --CF.sub.3, --OCH.sub.3,
nitro, or phosphate; R.sup.2 is halo, hydroxy, cyano, or nitro; q
is 0, 1 or 2; X is (CH.sub.2).sub.z; z is 1; R.sup.5, R.sup.6, le,
and R.sup.8 are H; Y is absent and W.sub.d is:
##STR00071## R.sup.11 is amino; and R.sup.12 is H, alkyl, alkynyl,
alkenyl, halo, aryl, heteroaryl, heterocycloalkyl, or
cycloalkyl.
In another aspect, R.sub.3 is H, CH.sub.3, CF.sub.3, Cl, or F; B is
aryl, heteroaryl, heterocycloalkyl, or cycloalkyl, R.sup.1 is H,
--F, --Cl, --CN, --CH.sub.3, isopropyl, --CF.sub.3, --OCH.sub.3,
nitro, or phosphate; R.sup.2 is halo, hydroxy, cyano, or nitro; q
is 0, 1 or 2; R.sup.5, R.sup.6, R.sup.7, and R.sup.8 are H; X is
(CH.sub.2).sub.z; z is 1; X is (CH.sub.2).sub.z; z is 1; Y is
--N(R.sup.9)--; R.sup.9 is hydrogen, methyl, or ethyl; and W.sub.d
is
##STR00072## In some embodiments, Y is --NH--.
In another aspect, for the compounds of Formula I R.sub.3 is aryl,
heteroaryl, H, CH.sub.3, CF.sub.3, Cl, or F; B is alkyl or a moiety
of Formula II;
wherein W.sub.c is aryl, heteroaryl, heterocycloalkyl, or
cycloalkyl, and q is an integer of 0, 1, 2, 3, or 4; R.sup.1 is H,
--F, --Cl, --CN, --CH.sub.3, isopropyl, --CF.sub.3, --OCH.sub.3,
nitro, or phosphate; R.sup.2 is halo, hydroxy, cyano, nitro, or
phosphate; q is 0, 1 or 2; R.sup.5, R.sup.6, R.sup.7, and R.sup.8
are H; X is absent or (CH(R.sup.9)).sub.z; z is an integer of 1, 2,
3, or 4; Y is absent, --N(R.sup.9)--, or --N(R.sup.9)
CH(R.sup.9)--; R.sup.9 is hydrogen, alkyl, cycloalkyl, or
heteroalkyl; at least one of X and Y is present; and W.sub.d is
pyrazolopyrimidine or purine. In some embodiments, when X is
present, Y is --N(R.sup.9)--, and W.sub.d is purine, then Y is
--NH--.
In another aspect, for the compounds of Formula I, R.sub.3 is aryl,
heteroaryl, H, CH.sub.3, CF.sub.3, Cl, or F; B is alkyl or a moiety
of Formula II which is aryl, heteroaryl, heterocycloalkyl, or
cycloalkyl, R.sup.1 is H, --F, --Cl, --CN, --CH.sub.3, isopropyl,
--CF.sub.3, --OCH.sub.3, nitro, or phosphate; R.sup.2 is halo,
hydroxy, cyano, nitro, or phosphate; q is 0, 1 or 2; R.sup.5,
R.sup.6, R.sup.7, and R.sup.8 are H; X is absent or
(CH(R.sup.9)).sub.z; z is an integer of 1, 2, 3, or 4; Y is absent,
--N(R.sup.9)--, or --N(R.sup.9) CH(R.sup.9)--; R.sup.9 is hydrogen,
methyl, or ethyl; at least one of X and Y is present; W.sub.d
is:
##STR00073## R.sup.11 is amino; and R.sup.12 is H, alkyl, alkynyl,
alkenyl, halo, aryl, heteroaryl, heterocycloalkyl, cycloalkyl,
cyano, amino, carboxylic acid, aloxycarbonyl, or amido. In some
embodiments, when X is present, Y is --N(R.sup.9)--, and W.sub.d is
purine, then Y is --NH--.
In another aspect, for the compounds of Formula I, R.sub.3 is H,
CH.sub.3, CF.sub.3, Cl, or F; B is alkyl or a moiety of Formula II
which is aryl, heteroaryl, heterocycloalkyl, or cycloalkyl, R.sup.1
is H, --F, --Cl, --CN, --CH.sub.3, isopropyl, --CF.sub.3,
--OCH.sub.3, nitro, or phosphate; R.sup.2 is halo, hydroxy, cyano,
nitro, or phosphate; q is 0, 1 or 2; R.sup.5, R.sup.6, R.sup.7, and
R.sup.8 are H; X is (CH(R.sup.9)).sub.z; z is an integer of 1; Y is
absent-; R.sup.9 is hydrogen, methyl, or ethyl; W.sub.d is:
##STR00074## R.sup.11 is amino; and R.sup.12 is H, alkyl, alkynyl,
alkenyl, halo, aryl, heteroaryl, heterocycloalkyl, cycloalkyl,
cyano, amino, carboxylic acid, alkoxycarbonyl, or amido.
In another aspect, for the compounds of Formula I, R.sub.3 is aryl,
heteroaryl, H, CH.sub.3, CF.sub.3, Cl, or F; B is a moiety of
Formula II which is aryl, heteroaryl, heterocycloalkyl, or
cycloalkyl, R.sup.1 is H, --F, --Cl, --CN, --CH.sub.3, isopropyl,
--CF.sub.3, --OCH.sub.3, nitro, or phosphate; R.sup.2 is halo,
hydroxy, cyano, nitro, or phosphate; q is 0, 1 or 2; R.sup.5,
R.sup.6, R.sup.7, and R.sup.8 are H; X is absent or
(CH(R.sup.9)).sub.z; z is an integer of 1; Y is absent,
--N(R.sup.9)--, or --N(R.sup.9) CH(R.sup.9)--; R.sup.9 is hydrogen,
methyl, or ethyl; at least one of X and Y is present, and W.sub.d
is:
##STR00075## In some embodiments, when X is present, Y is
--N(R.sup.9)--, and W.sub.d is purine, then Y is --NH--.
In another aspect, for the compounds of Formula I, R.sub.3 is aryl,
heteroaryl, H, CH.sub.3, CF.sub.3, Cl, or F; B is a moiety of
Formula II which is aryl, heteroaryl, heterocycloalkyl, or
cycloalkyl, R.sup.1 is H, --F, --Cl, --CN, --CH.sub.3, isopropyl,
--CF.sub.3, --OCH.sub.3, nitro, or phosphate; R.sup.2 is halo,
hydroxy, cyano, nitro, or phosphate; q is 0, 1 or 2; R.sup.5,
R.sup.6, R.sup.7, and R.sup.8 are H; X is absent; Y is --N(R.sup.9)
CH(R.sup.9)--; R.sup.9 is hydrogen, methyl, or ethyl; and W.sub.d
is:
##STR00076##
In another aspect, for the compounds of Formula I, R.sub.3 is aryl,
heteroaryl, H, CH.sub.3, CF.sub.3, Cl, or F; B is alkyl or a moiety
of Formula II which is aryl, heteroaryl, heterocycloalkyl, or
cycloalkyl, R.sup.1 is H, --F, --Cl, --CN, --CH.sub.3, isopropyl,
--CF.sub.3, --OCH.sub.3, nitro, or phosphate; R.sup.2 is halo,
hydroxy, cyano, nitro, or phosphate; q is 0, 1 or 2; R.sup.5,
R.sup.6, R.sup.7, and R.sup.8 are H; X is absent or
(CH(R.sup.9)).sub.z; z is an integer of 1, 2, 3, or 4; Y is absent,
--N(R.sup.9)--, or --N(R.sup.9) CH(R.sup.9)--; R.sup.9 is hydrogen,
methyl, or ethyl; at least one of X and Y is present; W.sub.d
is:
##STR00077## R.sup.a' is hydrogen, halo, or amino; and R.sup.12 is
H, H alkyl, alkynyl, alkenyl, halo, aryl, heteroaryl,
heterocycloalkyl, cycloalkyl, cyano, amino, carboxylic acid,
aloxycarbonyl, or amido. In some embodiments, when X is present, Y
is --N(R.sup.9)--, and W.sub.d is purine, then Y is --NH--.
Additional exemplary compounds of the present invention are
disclosed having a sub-structure of Formula IV-A.
##STR00078##
Some illustrative compounds of the present invention having a
structure of Formula IV-A include those in which R.sup.3 is --H,
--Cl, --F, or --CH.sub.3 in combination with any B moiety described
in Table 1, and any R.sup.12 as described in Table 2. A compound of
Formula IV-A includes any combination of R.sup.3, B, and R.sup.12.
Additional exemplary compounds of Formula IV-A are illustrated in
Table 4.
TABLE-US-00001 TABLE 1 Illustrative B moieties of the compounds of
Formula I. Sub- class # B B-1 ##STR00079## B-2 ##STR00080## B-3
--CH(CH.sub.3)2 B-4 ##STR00081## B-5 ##STR00082## B-6 ##STR00083##
B-7 ##STR00084## B-8 ##STR00085## B-9 ##STR00086## B-10
##STR00087## B-11 ##STR00088## B-12 ##STR00089## B-13 ##STR00090##
B-14 ##STR00091## B-15 ##STR00092## B-16 ##STR00093## B-17
##STR00094## B-18 ##STR00095## B-19 ##STR00096## B-20 ##STR00097##
B-21 ##STR00098## B-22 ##STR00099## B-23 ##STR00100## B-24
##STR00101## B-25 ##STR00102## B-26 ##STR00103## B-27 ##STR00104##
B-28 ##STR00105## B-29 ##STR00106## B-30 ##STR00107## B-31
##STR00108## B-32 ##STR00109## B-33 ##STR00110## B-34 ##STR00111##
B-35 ##STR00112## B-36 ##STR00113## B-37 ##STR00114## B-38
##STR00115## B-39 ##STR00116## B-40 ##STR00117## B-41 ##STR00118##
B-42 ##STR00119## B-43 ##STR00120## B-44 ##STR00121## B-45
##STR00122## B-46 ##STR00123## B-47 ##STR00124## B-48 ##STR00125##
B-49 ##STR00126## B-50 ##STR00127## B-51 ##STR00128## B-52
##STR00129## B-53 ##STR00130## B-54 ##STR00131## B-55 ##STR00132##
B-56 ##STR00133## B-57 ##STR00134## B-58 ##STR00135## B-59
##STR00136## B-60 ##STR00137## B-61 ##STR00138## B-62 ##STR00139##
B-63 ##STR00140## B-64 ##STR00141## B-65 ##STR00142## B-66
##STR00143## B-67 ##STR00144## B-68 ##STR00145## B-69 ##STR00146##
B-70 ##STR00147## B-71 ##STR00148## B-72 ##STR00149## B-73
##STR00150## B-74 ##STR00151## B-75 ##STR00152## B-76 ##STR00153##
B-77 ##STR00154## B-78 ##STR00155## B-79 ##STR00156## B-80
##STR00157## B-81 ##STR00158## B-82 ##STR00159## B-83 ##STR00160##
B-84 ##STR00161## B-85 ##STR00162## B-86 ##STR00163## B-87
--CH.sub.3 B-88 --CH.sub.2CH.sub.3 B-89 ##STR00164## B-90
##STR00165## B-91 ##STR00166## B-92 ##STR00167## B-93 ##STR00168##
B-94 ##STR00169## B-95 ##STR00170## B-96 ##STR00171## B-97
##STR00172## B-98 ##STR00173## B-99 ##STR00174## B-100 ##STR00175##
B-101 ##STR00176## B-102 ##STR00177##
TABLE-US-00002 TABLE 2 Illustrative R.sup.12 of compounds of
Formula I. Sub- class R.sup.12 12-1 --CN 12-2 --Br 12-3 --Cl 12-4
--CH.sub.2CH.sub.3 12-5 --CH.sub.3 12-6 --CH(CH.sub.3).sub.2 12-7
##STR00178## 12-8 ##STR00179## 12-9 ##STR00180## 12-10 ##STR00181##
12-11 ##STR00182## 12-12 ##STR00183## 12-13 ##STR00184## 12-14
##STR00185## 12-15 ##STR00186## 12-16 ##STR00187## 12-17
##STR00188## 12-18 ##STR00189## 12-19 ##STR00190## 12-20
##STR00191## 12-21 ##STR00192## 12-22 ##STR00193## 12-23
##STR00194## 12-24 ##STR00195## 12-25 ##STR00196## 12-26
##STR00197## 12-27 ##STR00198## 12-28 ##STR00199## 12-29
##STR00200## 12-30 ##STR00201## 12-31 ##STR00202## 12-32
##STR00203## 12-33 ##STR00204## 12-34 ##STR00205## 12-35 --H 12-36
##STR00206## 12-37 ##STR00207## 12-38 ##STR00208## 12-39
##STR00209## 12-40 ##STR00210## 12-41 ##STR00211## 12-42
##STR00212## 12-43 ##STR00213## 12-44 ##STR00214## 12-45
##STR00215## 12-46 ##STR00216## 12-47 ##STR00217## 12-48
##STR00218## 12-49 ##STR00219## 12-50 ##STR00220## 12-51
##STR00221## 12-52 ##STR00222## 12-53 ##STR00223## 12-54
##STR00224## 12-55 ##STR00225## 12-56 ##STR00226## 12-57
##STR00227## 12-58 ##STR00228## 12-59 ##STR00229## 12-60
##STR00230## 12-61 --I 12-62 ##STR00231## 12-63 ##STR00232## 12-64
##STR00233## 12-65 ##STR00234## 12-66 ##STR00235## 12-67
##STR00236## 12-68 ##STR00237## 12-69 ##STR00238## 12-70
##STR00239## 12-71 ##STR00240## 12-72 ##STR00241## 12-73
##STR00242## 12-74 ##STR00243## 12-75 ##STR00244## 12-76
##STR00245## 12-77 ##STR00246## 12-78 ##STR00247## 12-79
##STR00248## 12-80 ##STR00249## 12-81 ##STR00250## 12-82
##STR00251## 12-83 ##STR00252## 12-84 ##STR00253## 12-85
##STR00254## 12-86 ##STR00255## 12-87 ##STR00256## 12-88
##STR00257## 12-89 ##STR00258## 12-90 ##STR00259## 12-91
##STR00260## 12-92 ##STR00261## 12-93 ##STR00262## 12-94
##STR00263## 12-95 ##STR00264## 12-96 ##STR00265## 12-97 --F 12-98
##STR00266## 12-99 ##STR00267## 12-100 ##STR00268## 12-101
##STR00269## 12-102 ##STR00270##
Other illustrative compounds of the present invention have a
structure of Formula V-A, V-A1, or V-A2, wherein B is a moiety
described in Table 1, in combination with R.sup.3, which is --H,
--Cl, --F, or CH.sub.3, and R.sup.9, which is --H, --CH.sub.3, or
--CH.sub.2CH.sub.3. A compound of Formula V-A, V-A1, or V-A2
includes any combination of R.sup.3, B, and R.sup.9.
##STR00271##
Yet other illustrative compounds of the present invention have a
structure of Formula V-B, wherein B is a moiety described in Table
1, in combination with R.sup.3, which is --H, --Cl, --F, or
CH.sub.3, and R.sup.9, which is --H, --CH.sub.3, or
--CH.sub.2CH.sub.3. A compound of Formula V-B includes any
combination of R.sup.3, B, and R.sup.9.
##STR00272##
Some other illustrative compounds of the present invention have a
structure of Formula VI-A, wherein B is a moiety described in Table
1, in combination with R.sup.3, which is --H, --Cl, --F, or
CH.sub.3, and R.sup.9, which is --H, --CH.sub.3, or
--CH.sub.2CH.sub.3. A compound of Formula VI-A includes any
combination of R.sup.3, B, and R.sup.9.
##STR00273##
Further illustrative compounds of the invention have a structure of
one of Formulae VII-A1, VII-A2, VIII-A1, VIII-A2, IX-A1, IX-A2,
X-A1, X-A2, XI-A1, XI-A2, XII-A, XII-A1, XII-A2, XIII-A, XIII-A1,
XIII-A2, XIV-A, XIV-A1, or XIV-A2: wherein B is a moiety described
in Table 1, any R.sup.12 as described in Table 2, in combination
with R.sup.3, which is --H, --Cl, --F, or CH.sub.3, R.sup.9 which
is --H, --CH.sub.3, or --CH.sub.2CH.sub.3, and R.sup.a' which is
--H, --Cl, --F, or --NH.sub.2. A compound of Formulae VII-A1,
VII-A2, VIII-A1, VIII-A2, IX-A1, IX-A2, X-A1, X-A2, XI-A1, XI-A2,
XII-A, XII-A1, XII-A2, XIII-A, XIII-A1, XIII-A2, XIV-A, XIV-A1, or
XIV-A2: includes any combination of R.sup.a, R.sup.3, B, R.sup.9
and R.sup.12.
Additional exemplary compounds of the present invention include but
are not limited to the following:
##STR00274## ##STR00275## ##STR00276## ##STR00277## ##STR00278##
##STR00279## ##STR00280## ##STR00281## ##STR00282## ##STR00283##
##STR00284## ##STR00285## ##STR00286## ##STR00287## ##STR00288##
##STR00289##
The chemical entities described herein can be synthesized according
to one or more illustrative schemes herein and/or techniques well
known in the art.
Unless specified to the contrary, the reactions described herein
take place at atmospheric pressure, generally within a temperature
range from -10.degree. C. to 200.degree. C. Further, except as
otherwise specified, reaction times and conditions are intended to
be approximate, e.g., taking place at about atmospheric pressure
within a temperature range of about -10.degree. C. to about
110.degree. C. over a period of about 1 to about 24 hours;
reactions left to run overnight average a period of about 16
hours.
The terms "solvent," "organic solvent," and "inert solvent" each
mean a solvent inert under the conditions of the reaction being
described in conjunction therewith including, for example, benzene,
toluene, acetonitrile, tetrahydrofuran ("THF"), dimethylformamide
("DMF"), chloroform, methylene chloride (or dichloromethane),
diethyl ether, methanol, N-methylpyrrolidone ("NMP"), pyridine and
the like. Unless specified to the contrary, the solvents used in
the reactions described herein are inert organic solvents. Unless
specified to the contrary, for each gram of the limiting reagent,
one cc (or mL) of solvent constitutes a volume equivalent.
Isolation and purification of the chemical entities and
intermediates described herein can be effected, if desired, by any
suitable separation or purification procedure such as, for example,
filtration, extraction, crystallization, column chromatography,
thin-layer chromatography or thick-layer chromatography, or a
combination of these procedures. Specific illustrations of suitable
separation and isolation procedures can be had by reference to the
examples hereinbelow. However, other equivalent separation or
isolation procedures can also be used.
When desired, the (R)- and (S)-isomers of the compounds of the
present invention, if present, may be resolved by methods known to
those skilled in the art, for example by formation of
diastereoisomeric salts or complexes which may be separated, for
example, by crystallization; via formation of diastereoisomeric
derivatives which may be separated, for example, by
crystallization, gas-liquid or liquid chromatography; selective
reaction of one enantiomer with an enantiomer-specific reagent, for
example enzymatic oxidation or reduction, followed by separation of
the modified and unmodified enantiomers; or gas-liquid or liquid
chromatography in a chiral environment, for example on a chiral
support, such as silica with a bound chiral ligand or in the
presence of a chiral solvent. Alternatively, a specific enantiomer
may be synthesized by asymmetric synthesis using optically active
reagents, substrates, catalysts or solvents, or by converting one
enantiomer to the other by asymmetric transformation.
The compounds described herein can be optionally contacted with a
pharmaceutically acceptable acid to form the corresponding acid
addition salts.
Many of the optionally substituted starting compounds and other
reactants are commercially available, e.g., from Aldrich Chemical
Company (Milwaukee, Wis.) or can be readily prepared by those
skilled in the art using commonly employed synthetic
methodology.
The compounds of the invention can generally be synthesized by an
appropriate combination of generally well known synthetic methods.
Techniques useful in synthesizing these chemical entities are both
readily apparent and accessible to those of skill in the relevant
art, based on the instant disclosure.
The compounds of the invention can be synthesized by an appropriate
combination of known synthetic methods in the art. The discussion
below is offered to illustrate certain of the diverse methods
available for use in making the compounds of the invention and is
not intended to limit the scope of reactions or reaction sequences
that can be used in preparing the compounds of the present
invention.
##STR00290## ##STR00291##
Referring to Scheme 1, Step 1, a compound of Formula 101, wherein X
is N or CR.sup.7, is converted to a compound of Formula 103, for
example, via a two step process of Heck coupling with a compound of
Formula 102, followed by acid catalyzed cyclization in methanol.
The product, a compound of Formula 103, is isolated. Referring to
Scheme 1, Step 2, a compound of Formula 103 is converted to a
compound of Formula 404, for example, via reaction with an
appropriately substituted aniline. The product, a compound of
Formula 104, is isolated. Referring to Scheme 1, Step 3, a compound
of Formula 104 is converted to a compound of Formula 105, for
example, though reduction with lithium aluminum hydride. The
product, a compound of Formula 105, is isolated. Referring to
Scheme 1, Step 4, a compound of Formula 105 is converted to a
compound of Formula 106, for example, via reaction with thionyl
chloride. The product, a compound of Formula 106, is isolated.
Referring to Scheme 1, Step 5, a compound of Formula 106 is
converted to a compound of Formula 107, for example, via alkylation
with a pyrazolopyrimidine using a base such as potassium carbonate.
The product, a compound of Formula 107, is isolated. Referring to
Scheme 1, Step 6, a compound of Formula 107 is converted to a
compound of Formula 108, for example, via a Suzuki reaction. The
product, a compound of Formula 108, is isolated and optionally
purified.
##STR00292## ##STR00293##
Referring to Scheme 2, Step 1, a compound of Formula 201, wherein X
is N or CR.sup.7, is converted to a compound of Formula 202, for
example, with a reagent suitable for introduction of an acid
chloride, for example, oxalyl chloride. The product, a compound of
Formula 202, is optionally isolated. Referring to Scheme 2, Step 2,
a compound of Formula 202 is converted to a compound of Formula 503
for example, reaction with, for example, an aryl amine. The
product, a compound of Formula 203, is isolated. Referring to
Scheme 2, Step 3, a compound of Formula 203 is converted to a
compound of Formula 204, for example, via a Stille coupling using
an appropriate vinyl-stannane. The product, a compound of Formula
204, is isolated. Referring to Scheme 2, Step 4, a compound of
Formula 204 is converted to a tertiary amide, a compound of Formula
205, via reaction with chloroethyl acetate and sodium hydride base.
The compound of Formula 205 is isolated. Referring to Scheme 2,
Step 5, a compound of Formula 205 is oxidized to an aldehyde,
using, for example, osmonium tetraoxide and sodium periodinate. The
product, a compound of Formula 206, is isolated. Referring to
Scheme 2, Step 6, a compound of Formula 206 is converted to a
compound of Formula 104, for example, though aldol reaction in
ethanol with a base, such as cesium carbonate. The product, a
compound of Formula 104, is isolated. Referring to Scheme 2, Step
7, a compound of Formula 104 is reduced to a primary alcohol via
reduction with, for example, lithium aluminum hydride, to produce a
compound of Formula 105, which is isolated. Referring to Scheme 2,
Step 8, a compound of Formula 105 is converted to a compound of
Formula 207 via reaction with carbon tetrabromide and
triphenylphosphine. The compound of Formula 207 is isolated. This
compound can be a central intermediate in the synthesis of the
compounds of the invention.
##STR00294##
Referring to Scheme 3, Step 9, a compound of Formula 207, wherein X
is N or CR.sup.7, is synthesized as described in Reaction Scheme 2
and is converted to a compound of Formula 107 via coupling with the
compound of Formula 208 in the presence of base, for example,
potassium t-butoxide. The compound of Formula 107 is isolated.
Referring to Scheme 3, Step 10, a compound of Formula 107 is
converted to a compound of Formula 108 via coupling with, for
example, an aryl boronic acid, in the presence of coupling
catalysts and base, for example, palladium acetate,
triphenylphosphine and sodium carbonate, for example. The compound
of Formula 108 is isolated.
##STR00295##
##STR00296## ##STR00297##
Referring to Reaction Scheme 4A, which illustrates synthesis of a
general class of purinyl substituted isoquinolones, Step 1, iodo
ester 401, is reacted with an alkyne of Formula 400-A in the
presence of a palladium catalyst, copper iodide and triethylamine
(TEA) to couple the alkyne to the aryl core of compound 401 to
produce a compound of Formula 402. The compound of Formula 402 is
optionally isolated. Referring to Reaction Scheme 4, Step 2, a
compound of Formula 402 is treated with potassium hydroxide base to
obtain the carboxylic acid, a compound of Formula 403, if the
reaction product is acidified, or its salt. The compound of Formula
403 is optionally isolated. Referring to Reaction Scheme 4, Step 3,
a compound of Formula 403 is treated with
bis(acetonitrile)dichoropalladium (II) and TEA to effect
intramolecular ring closure to produce a compound of Formula 404.
The compound of Formula 404 is isolated. Referring to Reaction
Scheme 4, Step 4, a compound of Formula 404 is reacted with a
primary amine to produce a compound of Formula 405. The compound of
Formula 405 is optionally isolated. Referring to Reaction Scheme 4,
Step 5, a compound of Formula 405 is treated with hydrochoric acid,
removing the protecting group on nitrogen, and to obtain a compound
of Formula 406. The compound of Formula 406 is optionally isolated.
Referring to Reaction Scheme 4, Step 6, a compound of Formula 406
is reacted with a compound of Formula 407, to produce a compound of
Formula 408. The compound of Formula 408 is isolated.
In Reaction Scheme 4B, the synthesis of one subset of purinyl
substituted isoquinolones, wherein R.sup.9 is methyl and R.sup.a is
hydrogen, is illustrated using the synthetic transformations
described for Reaction Scheme 4A.
##STR00298##
Referring to Reaction Scheme 5, Step 1, iodo ester 401 is reacted
with alkyne 501 in the presence of palladium coupling catalyst,
copper iodide, and TEA, to obtain a compound of Formula 502. The
compound of Formula 502 is optionally isolated. Referring to
Reaction Scheme 5, Step 2, the compound of Formula 502 is treated
with potassium hydroxide base to obtain the carboxylate or free
acid of a compound of Formula 503. Referring to Reaction Scheme 5,
Step 3, the compound of Formula 503 is treated with
bis(acetonitrile)dichoropalladium (II) and TEA to effect
intramolecular ring closure to produce a compound of Formula 504.
The compound of Formula 504 is optionally isolated. Referring to
Reaction Scheme 5, Step 4, the compound of Formula 504 is treated
with a primary amine to produce a compound of Formula 505. The
compound of Formula 505 is isolated.
##STR00299## ##STR00300##
##STR00301## ##STR00302##
Referring to Reaction Scheme 6A, which illustrates synthesis of a
general class of purinyl substituted isoquinolones, Step 1, iodo
ester 401 is reacted with alkyne 601 in the presence of palladium
coupling catalyst, copper iodide, and TEA, to obtain a compound of
Formula 602. The compound of Formula 602 is optionally isolated.
Referring to Reaction Scheme 6, Step 2, the compound of Formula 602
is treated with potassium hydroxide base to obtain the carboxylate
or free acid of a compound of Formula 603. Referring to Reaction
Scheme 6, Step 3, the compound of Formula 603 is treated with
bis(acetonitrile)dichloropalladium (II) and TEA to effect
intramolecular ring closure to produce a compound of Formula 604.
The compound of Formula 604 is optionally isolated. Referring to
Reaction Scheme 6, Step 4, the compound of Formula 604 is treated
with a primary amine to produce a compound of Formula 605. The
compound of Formula 605 is isolated. Referring to Reaction Scheme
6, Step 5, the compound of Formula 605 is treated with acid to
remove the THP protecting group to obtain a compound of Formula
606. The compound of Formula 606 is isolated.
In Reaction Scheme 6B, the synthesis of purinyl substituted
isoquinolones, wherein R.sup.9 is methyl and R.sup.a is hydrogen,
is illustrated using the synthetic transformations described for
Reaction Scheme 6A.
##STR00303##
##STR00304##
Referring to Reaction Scheme 7A, which illustrates the synthesis of
purinyl or pyrazolopyrimidinyl substituted isoquinoliones
comprising an alkyl amine substituent at the position represented
by B in Formula I, Step 1 the compound of Formula 701 is
synthesized by a variety of synthetic routes, including variations
of Schemes 1 or 2 where, for example, a benzyl amine is used in the
step of converting a compound of Formula 103 to a compound of
Formula 104. The benzyl protecting group of the amine may be
removed by standard deprotection chemistry to produce a compound of
701. Another example of a conversion of a compound of Formula 103
to a compound of Formula 701, treatment of the compound of Formula
103 with ammonia produces the compound of Formula 701. The compound
of Formula 701 is converted to a compound of Formula 702 by
alkylation of the amide nitrogen with a number of 2-carbon
containing synthons which can be deprotected, oxidized and
reprotected as the respective ketal, the compound of Formula 702.
Referring to Reaction Scheme 7, Step 2-1, the compound of Formula
702 is transformed by, for example, reductive amination of the
ester moiety to introduce the purinyl moiety of a compound of
Formula 703, or alternatively, is alkylated to so introduce a
purinyl moiety and obtain a compound of Formula 703. Referring to
Reaction Scheme 7, Step 3-1, the compound of Formula 703 is treated
with acid to remove the ketal protecting group to produce a
compound of Formula 704. The compound of Formula 704 is isolated.
Referring to Reaction Scheme 7, Step 4-1, the compound of Formula
704 is reductively aminated with an amine to produce a compound of
Formula 705. The compound of Formula 705 is isolated. Referring to
Reaction Scheme 7, Step 2-2, the compound of Formula 702 is
transformed by, steps 7 and 8 of Scheme 2 and step 9 of Scheme 3 to
introduce the pyrazolopyrimidine moiety of a compound of Formula
706. The compound of Formula 706 is isolated. Referring to Reaction
Scheme 7, Step 3-2, the compound of Formula 706 is treated with
acid to remove the ketal protecting group to produce a compound of
Formula 707. The compound of Formula 707 is isolated.
Referring to Reaction Scheme 7, Step 4-2, the compound of Formula
707 is reductively aminated with an amine to produce a compound of
Formula 708. The compound of Formula 708 is isolated.
In Reaction Scheme 7B, the synthesis of compounds wherein R.sup.9
is methyl and R.sup.a is hydrogen is illustrated, using the steps
described in Scheme 7A.
##STR00305##
Referring to Reaction Scheme 8, Step 1, the compound of Formula 701
is synthesized as described in Scheme 7 or any other generally
known chemistry. The compound of Formula 701 is transformed by
alkylation of the amide nitrogen with a number of 2-carbon
containing synthons which can be deprotected, and converted to the
alkoxy protected species as shown in the compound of Formula 801,
which can be isolated. Referring to Reaction Scheme 8, Step 2, the
compound of Formula 801 is converted via chemistry described in
Step 2-1 of Scheme 7 to introduce a purinyl moiety, and that
resultant compound is transformed by deprotection, activation and
amination with an amine to produce a compound of Formula 802, which
is isolated.
Referring to Reaction Scheme 8, Step 3, the compound of Formula 801
is converted via chemistry described in Step 2-2 of Scheme 7 to
introduce a pyrazolopyrimidine moiety, and that resultant compound
is transformed by deprotection, activation and amination with an
amine to produce a compound of Formula 803, which is isolated.
##STR00306##
Referring to Reaction Scheme 9, Step 1, the compound of Formula 901
is treated with an amine to produce a compound of Formula 902. The
compound of Formula 902 is isolated. Referring to Reaction Scheme
9, Step 2, the compound of Formula 902 is treated with phosphorus
oxychloride to generate a compound of Formula 903. The compound of
Formula 903 is isolated. Referring to Reaction Scheme 9, Step 3,
the compound of Formula 903 is reacted with an amino purine of
Formula 904 to obtain a compound of Formula 905. The compound of
Formula 905 is isolated. Referring to Reaction Scheme 9, Step 4,
the compound of Formula 905 is treated with hydrochloric acid to
remove the protecting group at nitrogen on the purine moiety to
produce a compound of Formula 906. The compound of 906 is
isolated.
##STR00307##
Referring to Reaction Scheme 10, Step 1, the compound of Formula
1001 is treated with vinylogous ester 1002 using, for example a
Heck reaction with subsequent cyclization, to produce a compound of
Formula 1003. The compound of Formula 1003 is isolated. Referring
to Reaction Scheme 10, Step 2, the compound of Formula 1003 is
reacted with 4-amino N-Boc piperidine to produce a compound of
Formula 1004. The compound of Formula 1004 is isolated. The
compound of Formula 1004 can be used as an intermediate in the
synthesis of the compounds of the invention.
##STR00308##
Referring to Reaction Scheme 11, Step 1, the compound of Formula
1101 is treated with an alkynyl alcohol, for example, of Formula
1102, in the presence of copper iodide and palladium on carbon
catalyst, to produce a compound of Formula 1103. The compound of
Formula 1103 is isolated. Referring to Reaction Scheme 11, Step 1,
the compound of Formula 1102 is reacted with 4-amino N-Boc
piperidine to produce a compound of Formula 1103. The compound of
Formula 1103 is isolated. The compound of Formula 1103 can be used
as an intermediate in the synthesis of the compounds of the
invention.
##STR00309##
Another approach to synthesis of compounds of Formula I is
illustrated in Scheme 12. Referring to Step 1, the compound of
Formula 1201 is treated with a chlorinating agent such as oxalyl
chloride to produce an acid chloride of Formula 1202. In Step 2,
the compound of Formula 1202 is reacted with a compound of Formula
R'NH.sub.2 in the presence of a base such as triethylamine, to
produce a compound of Formula 1203. In Step 3, the compound of
Formula 1203 is treated with n-butyllithium and then reacted with
an dialkyl oxalate such as diethyl oxalate to produce a compound of
Formula 1204. In Step 4, the compound of Formula 1204 is refluxed
in an acidic solution, for example, hydrochloric acid in methanol
to produce a compound of Formula 1205. In Step 5, the compound of
Formula 1205 is treated with a reducing agent such as lithium
aluminum hydride to produce a compound of Formula 1206. In Step 6,
the compound of Formula 1206 is reacted with a brominating agent
such as phosphorus tribromide, in the presence of dimethylformamide
in acetonitrile to produce a bromo compound of Formula 1207. In
Step 7, the compound of Formula 1207 is reacted with a heteroaryl
compound, for example 3-iodo 1H-pyrazolo[3,4-d]pyrimidin-4-amine,
in the presence of a base such as potassium tert-butoxide in
dimethylformamide to produce a compound of Formula 1208.
##STR00310##
In Scheme 13, an approach is described for synthesizing compounds
of Formula I having a XY linker wherein X is predominately or
solely (S)--C(CH.sub.3)H-- and Y is --NH--. Wd is a monocyclic or
bicyclic heteroaryl, including but not limited to purinyl,
pyrimidinyl, pyrrolopyrimidinyl, or pyrazolopyrimidinyl. Referring
to Step 1 of Scheme 13, the compound of Formula 1301, (the
S-isomer) is coupled to N,O-dimethylhydroxylamine using
hydroxybenzotriazole (HOBt) and
1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide (EDCI) in the
presence of triethylamine to produce a compound of Formula 1302. In
Step 2, a compound of Formula 1203, which may be synthesized as
described in Scheme 12, is deprotonated with n-butyllithium in THF
and hexamethylphosphoramide at -78.degree. C. under an argon
atmosphere. The compound of Formula 1302 is added and the reaction
mixture is allowed to warm to -50.degree. C., quenched with the
addition of water and a compound of Formula 1303 is isolated. In
Step 3 the compound of Formula 1303 is treated with hydrochloric
acid in methanol at reflux, and then the reaction mixture is
basified with the addition of sodium carbonate solution to a pH of
about 7- about 8, to produce a compound of Formula 1304. The
compound of formula 1304 may be partly epimerized as a result of
the preceding reaction steps. Highly enantiopure 1304 may be
isolated by preparing the tartaric acid salt by dissolving the
compound of Formula 1304 in methanol and adding D-tartaric acid.
The resulting reaction mixture is refluxed for one hour, then
stirred at room temperature for 16 hours, and permits isolation of
the salt of the compound of Formula 1304 wherein the enantiomeric
purity is greater than 90% of the (S)-isomer. The free amine of the
compound of Formula 1304 is regenerated before its use in the next
synthesis step. The compound of Formula 1304, which is
substantially the (S)-enantiomer is coupled to a chloro substituted
heteroaryl W.sub.d, a compound of Formula 1305, including but not
limited to 6-chloro-9(tetrahydro-2H-pyran-2-yl)-9H-purine,
2,4,5,-trichloropyrimidine, 4-chloro-7H-pyrrolo[2,3-d]pyrimidine,
and 4-chloro-1H-pyrazolo[3,4-d]pyrimidine in the presence of base
such as diisopropylethylamine or ammonia, to produce a compound of
Formula 1306, and where the compound of Formula 1306 is the
(S)-isomer.
Synthesis of R.sup.3-Halo Analogs, e.g. Chloro Substituted
Isoquinolone Analogs.
The same reaction scheme 13 applies to the generation of a compound
having the formula:
##STR00311## wherein R.sup.3 is chloro.
Compounds disclosed herein can be synthesized using the reaction
schemes as disclosed herein, variants thereof, or other synthetic
methods known in the art.
In some embodiments, the compounds of the present invention
exhibits one or more functional characteristics disclosed herein.
For example, one or more subject compounds bind specifically to a
PI3 kinase. In some embodiments, the IC50 of a subject compound for
p110.alpha., p110.beta., p110.gamma., or p110.delta. is less than
about 1 uM, less than about 100 nM, less than about 50 nM, less
than about 10 nM, less than about 1 nM, less than about 0.5 nM,
less than about 100 pM, or less than about 50 pM.
In some embodiments, one or more of the subject compounds may
selectively inhibit one or more members of type I or class I
phosphatidylinositol 3-kinases (PI3-kinase) with an IC50 value of
about 100 nM, 50 nM, 10 nM, 5 nM, 100 pM, 10 pM or 1 pM, or less as
measured in an in vitro kinase assay.
Additionally, a compound of Formula having an (S)-isomeric
configuration with respect to carbon X may exhibit greater potency
against one or more target PI3-kinases than the corresponding
compound having an (R)-isomeric configuration with respect to
carbon X. For example, the compound of Formula I having an
(S)-isomeric configuration with respect to carbon X may have a
PI3-kinase IC50 value which is 1, 2, 3, or 4 orders of magnitude
lower than the PI3-kinase IC50 value of the corresponding compound
having an (R)-configuration.
In some embodiments, one or more of the subject compound may
selectively inhibit one or two members of type I or class I
phosphatidylinositol 3-kinases (PI3-kinase) consisting of
PI3-kinase .alpha., PI3-kinase .beta., PI3-kinase .gamma., and
PI3-kinase .delta.. In some aspects, some of the subject compounds
selectively inhibit PI3-kinase .delta. as compared to all other
type I PI3-kinases. In other aspects, some of the subject compounds
selectively inhibit PI3-kinase .delta. and PI3-kinase .gamma. as
compared to the rest of the type I PI3-kinases. In yet other
aspects, some of the subject compounds selectively inhibit
PI3-kinase .alpha. and PI3-kinase .beta. as compared to the rest of
the type I PI3-kinases. In still yet some other aspects, some of
the subject compounds selectively inhibit PI3-kinase .delta. and
PI3-kinase .alpha. as compared to the rest of the type I
PI3-kinases. In still yet some other aspects, some of the subject
compounds selectively inhibit PI3-kinase .delta. and PI3-kinase as
compared to the rest of the type I PI3-kinases, or selectively
inhibit PI3-kinase .delta. and PI3-kinase .alpha. as compared to
the rest of the type I PI3-kinases, or selectively inhibit
PI3-kinase .alpha. and PI3-kinase .gamma. as compared to the rest
of the type I PI3-kinases, or selectively inhibit PI3-kinase
.gamma. and PI3-kinase as compared to the rest of the type I
PI3-kinases.
In yet another aspect, an inhibitor that selectively inhibits one
or more members of type I PI3-kinases, or an inhibitor that
selectively inhibits one or more type I PI3-kinase mediated
signaling pathways, alternatively can be understood to refer to a
compound that exhibits a 50% inhibitory concentration (IC50) with
respect to a given type I PI3-kinase, that is at least at least
10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at
least 1000-fold, at least 10,100-fold, or lower, than the
inhibitor's IC50 with respect to the rest of the other type I
PI3-kinases.
Pharmaceutical Compositions
The invention provides pharmaceutical compositions comprising one
or more compounds of the present invention.
In some embodiments, the invention provides pharmaceutical
compositions for treating diseases or conditions related to an
undesirable, over-active, harmful or deleterious immune response in
a mammal. Such undesirable immune response can be associated with
or result in, e.g., asthma, emphysema, bronchitis, psoriasis,
allergy, anaphylaxis, auto-immune diseases, rheumatoid arthritis,
graft versus host disease, and lupus erythematosus. The
pharmaceutical compositions of the present invention can be used to
treat other respiratory diseases including but not limited to
diseases affecting the lobes of lung, pleural cavity, bronchial
tubes, trachea, upper respiratory tract, or the nerves and muscle
for breathing.
In some embodiments, the invention provides pharmaceutical
compositions for the treatment of disorders such as
hyperproliferative disorder including but not limited to cancer
such as acute myeloid leukemia, thymus, brain, lung, squamous cell,
skin, eye, retinoblastoma, intraocular melanoma, oral cavity and
oropharyngeal, bladder, gastric, stomach, pancreatic, bladder,
breast, cervical, head, neck, renal, kidney, liver, ovarian,
prostate, colorectal, esophageal, testicular, gynecological,
thyroid, CNS, PNS, AIDS related AIDS-Related (e.g. Lymphoma and
Kaposi's Sarcoma) or Viral-Induced cancer. In some embodiments,
said pharmaceutical composition is for the treatment of a
non-cancerous hyperproliferative disorder such as benign
hyperplasia of the skin (e. g., psoriasis), restenosis, or prostate
(e. g., benign prostatic hypertrophy (BPH)).
The invention also provides compositions for the treatment of liver
diseases (including diabetes), pancreatitis or kidney disease
(including proliferative glomerulonephritis and diabetes-induced
renal disease) or pain in a mammal.
The invention further provides a composition for the prevention of
blastocyte implantation in a mammal.
The invention also relates to a composition for treating a disease
related to vasculogenesis or angiogenesis in a mammal which can
manifest as tumor angiogenesis, chronic inflammatory disease such
as rheumatoid arthritis, inflammatory bowel disease,
atherosclerosis, skin diseases such as psoriasis, eczema, and
scleroderma, diabetes, diabetic retinopathy, retinopathy of
prematurity, age-related macular degeneration, hemangioma, glioma,
melanoma, Kaposi's sarcoma and ovarian, breast, lung, pancreatic,
prostate, colon and epidermoid cancer.
The subject pharmaceutical compositions are typically formulated to
provide a therapeutically effective amount of a compound of the
present invention as the active ingredient, or a pharmaceutically
acceptable salt, ester, prodrug, solvate, hydrate or derivative
thereof. Where desired, the pharmaceutical compositions contain
pharmaceutically acceptable salt and/or coordination complex
thereof, and one or more pharmaceutically acceptable excipients,
carriers, including inert solid diluents and fillers, diluents,
including sterile aqueous solution and various organic solvents,
permeation enhancers, solubilizers and adjuvants.
The subject pharmaceutical compositions can be administered alone
or in combination with one or more other agents, which are also
typically administered in the form of pharmaceutical compositions.
Where desired, the subject compounds and other agent(s) may be
mixed into a preparation or both components may be formulated into
separate preparations to use them in combination separately or at
the same time.
In some embodiments, the concentration of one or more of the
compounds provided in the pharmaceutical compositions of the
present invention is less than 100%, 90%, 80%, 70%, 60%, 50%, 40%,
30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%,
7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%,
0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%,
0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%,
0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%,
0.0002%, or 0.0001% w/w, w/v or v/v.
In some embodiments, the concentration of one or more of the
compounds of the present invention is greater than 90%, 80%, 70%,
60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25% 19%, 18.75%,
18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%, 16.75%, 16.50%,
16.25% 16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25% 14%,
13.75%, 13.50%, 13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%,
11.50%, 11.25% 11%, 10.75%, 10.50%, 10.25% 10%, 9.75%, 9.50%, 9.25%
9%, 8.75%, 8.50%, 8.25% 8%, 7.75%, 7.50%, 7.25% 7%, 6.75%, 6.50%,
6.25% 6%, 5.75%, 5.50%, 5.25% 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%,
3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%,
0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%,
0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%,
0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%,
0.0005%, 0.0004%, 0.0003%, 0.0002%, or 0.0001% w/w, w/v, or
v/v.
In some embodiments, the concentration of one or more of the
compounds of the present invention is in the range from
approximately 0.0001% to approximately 50%, approximately 0.001% to
approximately 40%, approximately 0.01% to approximately 30%,
approximately 0.02% to approximately 29%, approximately 0.03% to
approximately 28%, approximately 0.04% to approximately 27%,
approximately 0.05% to approximately 26%, approximately 0.06% to
approximately 25%, approximately 0.07% to approximately 24%,
approximately 0.08% to approximately 23%, approximately 0.09% to
approximately 22%, approximately 0.1% to approximately 21%,
approximately 0.2% to approximately 20%, approximately 0.3% to
approximately 19%, approximately 0.4% to approximately 18%,
approximately 0.5% to approximately 17%, approximately 0.6% to
approximately 16%, approximately 0.7% to approximately 15%,
approximately 0.8% to approximately 14%, approximately 0.9% to
approximately 12%, approximately 1% to approximately 10% w/w, w/v
or v/v. v/v.
In some embodiments, the concentration of one or more of the
compounds of the present invention is in the range from
approximately 0.001% to approximately 10%, approximately 0.01% to
approximately 5%, approximately 0.02% to approximately 4.5%,
approximately 0.03% to approximately 4%, approximately 0.04% to
approximately 3.5%, approximately 0.05% to approximately 3%,
approximately 0.06% to approximately 2.5%, approximately 0.07% to
approximately 2%, approximately 0.08% to approximately 1.5%,
approximately 0.09% to approximately 1%, approximately 0.1% to
approximately 0.9% w/w, w/v or v/v.
In some embodiments, the amount of one or more of the compounds of
the present invention is equal to or less than 10 g, 9.5 g, 9.0 g,
8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g, 4.5 g, 4.0
g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g,
0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g,
0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07
g, 0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008
g, 0.007 g, 0.006 g, 0.005 g, 0.004 g, 0.003 g, 0.002 g, 0.001 g,
0.0009 g, 0.0008 g, 0.0007 g, 0.0006 g, 0.0005 g, 0.0004 g, 0.0003
g, 0.0002 g, or 0.0001 g.
In some embodiments, the amount of one or more of the compounds of
the present invention is more than 0.0001 g, 0.0002 g, 0.0003 g,
0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 0.0008 g, 0.0009 g, 0.001
g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g, 0.004 g, 0.0045
g, 0.005 g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g, 0.008
g, 0.0085 g, 0.009 g, 0.0095 g, 0.01 g, 0.015 g, 0.02 g, 0.025 g,
0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g,
0.07 g, 0.075 g, 0.08 g, 0.085 g, 0.09 g, 0.095 g, 0.1 g, 0.15 g,
0.2 g, 0.25 g, 0.3 g, 0.35 g, 0.4 g, 0.45 g, 0.5 g, 0.55 g, 0.6 g,
0.65 g, 0.7 g, 0.75 g, 0.8 g, 0.85 g, 0.9 g, 0.95 g, 1 g, 1.5 g, 2
g, 2.5, 3 g, 3.5, 4 g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8
g, 8.5 g, 9 g, 9.5 g, or 10 g.
In some embodiments, the amount of one or more of the compounds of
the present invention is in the range of 0.0001-10 g, 0.0005-9 g,
0.001-8 g, 0.005-7 g, 0.01-6 g, 0.05-5 g, 0.1-4 g, 0.5-4 g, or 1-3
g.
The compounds according to the invention are effective over a wide
dosage range. For example, in the treatment of adult humans,
dosages from 0.01 to 1000 mg, from 0.5 to 100 mg, from 1 to 50 mg
per day, and from 5 to 40 mg per day are examples of dosages that
may be used. An exemplary dosage is 10 to 30 mg per day. The exact
dosage will depend upon the route of administration, the form in
which the compound is administered, the subject to be treated, the
body weight of the subject to be treated, and the preference and
experience of the attending physician.
Described below are non-limiting exemplary pharmaceutical
compositions and methods for preparing the same.
Pharmaceutical Compositions for Oral Administration
In some embodiments, the invention provides a pharmaceutical
composition for oral administration containing a compound of the
present invention, and a pharmaceutical excipient suitable for oral
administration.
In some embodiments, the invention provides a solid pharmaceutical
composition for oral administration containing: (i) an effective
amount of a compound of the present invention; optionally (ii) an
effective amount of a second agent; and (iii) a pharmaceutical
excipient suitable for oral administration. In some embodiments,
the composition further contains: (iv) an effective amount of a
third agent.
In some embodiments, the pharmaceutical composition may be a liquid
pharmaceutical composition suitable for oral consumption.
Pharmaceutical compositions of the invention suitable for oral
administration can be presented as discrete dosage forms, such as
capsules, cachets, or tablets, or liquids or aerosol sprays each
containing a predetermined amount of an active ingredient as a
powder or in granules, a solution, or a suspension in an aqueous or
non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil
liquid emulsion. Such dosage forms can be prepared by any of the
methods of pharmacy, but all methods include the step of bringing
the active ingredient into association with the carrier, which
constitutes one or more necessary ingredients. In general, the
compositions are prepared by uniformly and intimately admixing the
active ingredient with liquid carriers or finely divided solid
carriers or both, and then, if necessary, shaping the product into
the desired presentation. For example, a tablet can be prepared by
compression or molding, optionally with one or more accessory
ingredients. Compressed tablets can be prepared by compressing in a
suitable machine the active ingredient in a free-flowing form such
as powder or granules, optionally mixed with an excipient such as,
but not limited to, a binder, a lubricant, an inert diluent, and/or
a surface active or dispersing agent. Molded tablets can be made by
molding in a suitable machine a mixture of the powdered compound
moistened with an inert liquid diluent.
This invention further encompasses anhydrous pharmaceutical
compositions and dosage forms comprising an active ingredient,
since water can facilitate the degradation of some compounds. For
example, water may be added (e.g., 5%) in the pharmaceutical arts
as a means of simulating long-term storage in order to determine
characteristics such as shelf-life or the stability of formulations
over time. Anhydrous pharmaceutical compositions and dosage forms
of the invention can be prepared using anhydrous or low moisture
containing ingredients and low moisture or low humidity conditions.
Pharmaceutical compositions and dosage forms of the invention which
contain lactose can be made anhydrous if substantial contact with
moisture and/or humidity during manufacturing, packaging, and/or
storage is expected. An anhydrous pharmaceutical composition may be
prepared and stored such that its anhydrous nature is maintained.
Accordingly, anhydrous compositions may be packaged using materials
known to prevent exposure to water such that they can be included
in suitable formulary kits. Examples of suitable packaging include,
but are not limited to, hermetically sealed foils, plastic or the
like, unit dose containers, blister packs, and strip packs.
An active ingredient can be combined in an intimate admixture with
a pharmaceutical carrier according to conventional pharmaceutical
compounding techniques. The carrier can take a wide variety of
forms depending on the form of preparation desired for
administration. In preparing the compositions for an oral dosage
form, any of the usual pharmaceutical media can be employed as
carriers, such as, for example, water, glycols, oils, alcohols,
flavoring agents, preservatives, coloring agents, and the like in
the case of oral liquid preparations (such as suspensions,
solutions, and elixirs) or aerosols; or carriers such as starches,
sugars, micro-crystalline cellulose, diluents, granulating agents,
lubricants, binders, and disintegrating agents can be used in the
case of oral solid preparations, in some embodiments without
employing the use of lactose. For example, suitable carriers
include powders, capsules, and tablets, with the solid oral
preparations. If desired, tablets can be coated by standard aqueous
or nonaqueous techniques.
Binders suitable for use in pharmaceutical compositions and dosage
forms include, but are not limited to, corn starch, potato starch,
or other starches, gelatin, natural and synthetic gums such as
acacia, sodium alginate, alginic acid, other alginates, powdered
tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl
cellulose, cellulose acetate, carboxymethyl cellulose calcium,
sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl
cellulose, pre-gelatinized starch, hydroxypropyl methyl cellulose,
microcrystalline cellulose, and mixtures thereof.
Examples of suitable fillers for use in the pharmaceutical
compositions and dosage forms disclosed herein include, but are not
limited to, talc, calcium carbonate (e.g., granules or powder),
microcrystalline cellulose, powdered cellulose, dextrates, kaolin,
mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch,
and mixtures thereof.
Disintegrants may be used in the compositions of the invention to
provide tablets that disintegrate when exposed to an aqueous
environment. Too much of a disintegrant may produce tablets which
may disintegrate in the bottle. Too little may be insufficient for
disintegration to occur and may thus alter the rate and extent of
release of the active ingredient(s) from the dosage form. Thus, a
sufficient amount of disintegrant that is neither too little nor
too much to detrimentally alter the release of the active
ingredient(s) may be used to form the dosage forms of the compounds
disclosed herein. The amount of disintegrant used may vary based
upon the type of formulation and mode of administration, and may be
readily discernible to those of ordinary skill in the art. About
0.5 to about 15 weight percent of disintegrant, or about 1 to about
5 weight percent of disintegrant, may be used in the pharmaceutical
composition. Disintegrants that can be used to form pharmaceutical
compositions and dosage forms of the invention include, but are not
limited to, agar-agar, alginic acid, calcium carbonate,
microcrystalline cellulose, croscarmellose sodium, crospovidone,
polacrilin potassium, sodium starch glycolate, potato or tapioca
starch, other starches, pre-gelatinized starch, other starches,
clays, other algins, other celluloses, gums or mixtures
thereof.
Lubricants which can be used to form pharmaceutical compositions
and dosage forms of the invention include, but are not limited to,
calcium stearate, magnesium stearate, mineral oil, light mineral
oil, glycerin, sorbitol, mannitol, polyethylene glycol, other
glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated
vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil,
sesame oil, olive oil, corn oil, and soybean oil), zinc stearate,
ethyl oleate, ethylaureate, agar, or mixtures thereof. Additional
lubricants include, for example, a syloid silica gel, a coagulated
aerosol of synthetic silica, or mixtures thereof. A lubricant can
optionally be added, in an amount of less than about 1 weight
percent of the pharmaceutical composition.
When aqueous suspensions and/or elixirs are desired for oral
administration, the essential active ingredient therein may be
combined with various sweetening or flavoring agents, coloring
matter or dyes and, if so desired, emulsifying and/or suspending
agents, together with such diluents as water, ethanol, propylene
glycol, glycerin and various combinations thereof.
The tablets can be uncoated or coated by known techniques to delay
disintegration and absorption in the gastrointestinal tract and
thereby provide a sustained action over a longer period. For
example, a time delay material such as glyceryl monostearate or
glyceryl distearate can be employed. Formulations for oral use can
also be presented as hard gelatin capsules wherein the active
ingredient is mixed with an inert solid diluent, for example,
calcium carbonate, calcium phosphate or kaolin, or as soft gelatin
capsules wherein the active ingredient is mixed with water or an
oil medium, for example, peanut oil, liquid paraffin or olive
oil.
Surfactant which can be used to form pharmaceutical compositions
and dosage forms of the invention include, but are not limited to,
hydrophilic surfactants, lipophilic surfactants, and mixtures
thereof. That is, a mixture of hydrophilic surfactants may be
employed, a mixture of lipophilic surfactants may be employed, or a
mixture of at least one hydrophilic surfactant and at least one
lipophilic surfactant may be employed.
A suitable hydrophilic surfactant may generally have an HLB value
of at least 10, while suitable lipophilic surfactants may generally
have an HLB value of or less than about 10. An empirical parameter
used to characterize the relative hydrophilicity and hydrophobicity
of non-ionic amphiphilic compounds is the hydrophilic-lipophilic
balance ("HLB" value). Surfactants with lower HLB values are more
lipophilic or hydrophobic, and have greater solubility in oils,
while surfactants with higher HLB values are more hydrophilic, and
have greater solubility in aqueous solutions. Hydrophilic
surfactants are generally considered to be those compounds having
an HLB value greater than about 10, as well as anionic, cationic,
or zwitterionic compounds for which the HLB scale is not generally
applicable. Similarly, lipophilic (i.e., hydrophobic) surfactants
are compounds having an HLB value equal to or less than about 10.
However, HLB value of a surfactant is merely a rough guide
generally used to enable formulation of industrial, pharmaceutical
and cosmetic emulsions.
Hydrophilic surfactants may be either ionic or non-ionic. Suitable
ionic surfactants include, but are not limited to, alkylammonium
salts; fusidic acid salts; fatty acid derivatives of amino acids,
oligopeptides, and polypeptides; glyceride derivatives of amino
acids, oligopeptides, and polypeptides; lecithins and hydrogenated
lecithins; lysolecithins and hydrogenated lysolecithins;
phospholipids and derivatives thereof; lysophospholipids and
derivatives thereof; carnitine fatty acid ester salts; salts of
alkylsulfates; fatty acid salts; sodium docusate; acylactylates;
mono- and di-acetylated tartaric acid esters of mono- and
di-glycerides; succinylated mono- and di-glycerides; citric acid
esters of mono- and di-glycerides; and mixtures thereof.
Within the aforementioned group, ionic surfactants include, by way
of example: lecithins, lysolecithin, phospholipids,
lysophospholipids and derivatives thereof; carnitine fatty acid
ester salts; salts of alkylsulfates; fatty acid salts; sodium
docusate; acylactylates; mono- and di-acetylated tartaric acid
esters of mono- and di-glycerides; succinylated mono- and
di-glycerides; citric acid esters of mono- and di-glycerides; and
mixtures thereof.
Ionic surfactants may be the ionized forms of lecithin,
lysolecithin, phosphatidylcholine, phosphatidylethanolamine,
phosphatidylglycerol, phosphatidic acid, phosphatidylserine,
lysophosphatidylcholine, lysophosphatidylethanolamine,
lysophosphatidylglycerol, lysophosphatidic acid,
lysophosphatidylserine, PEG-phosphatidylethanolamine,
PVP-phosphatidylethanolamine, lactylic esters of fatty acids,
stearoyl-2-lactylate, stearoyl lactylate, succinylated
monoglycerides, mono/diacetylated tartaric acid esters of
mono/diglycerides, citric acid esters of mono/diglycerides,
cholylsarcosine, caproate, caprylate, caprate, laurate, myristate,
palmitate, oleate, ricinoleate, linoleate, linolenate, stearate,
lauryl sulfate, teracecyl sulfate, docusate, lauroyl carnitines,
palmitoyl carnitines, myristoyl carnitines, and salts and mixtures
thereof.
Hydrophilic non-ionic surfactants may include, but not limited to,
alkylglucosides; alkylmaltosides; alkylthioglucosides; lauryl
macrogolglycerides; polyoxyalkylene alkyl ethers such as
polyethylene glycol alkyl ethers; polyoxyalkylene alkylphenols such
as polyethylene glycol alkyl phenols; polyoxyalkylene alkyl phenol
fatty acid esters such as polyethylene glycol fatty acids
monoesters and polyethylene glycol fatty acids diesters;
polyethylene glycol glycerol fatty acid esters; polyglycerol fatty
acid esters; polyoxyalkylene sorbitan fatty acid esters such as
polyethylene glycol sorbitan fatty acid esters; hydrophilic
transesterification products of a polyol with at least one member
of the group consisting of glycerides, vegetable oils, hydrogenated
vegetable oils, fatty acids, and sterols; polyoxyethylene sterols,
derivatives, and analogues thereof; polyoxyethylated vitamins and
derivatives thereof; polyoxyethylene-polyoxypropylene block
copolymers; and mixtures thereof; polyethylene glycol sorbitan
fatty acid esters and hydrophilic transesterification products of a
polyol with at least one member of the group consisting of
triglycerides, vegetable oils, and hydrogenated vegetable oils. The
polyol may be glycerol, ethylene glycol, polyethylene glycol,
sorbitol, propylene glycol, pentaerythritol, or a saccharide.
Other hydrophilic-non-ionic surfactants include, without
limitation, PEG-10 laurate, PEG-12 laurate, PEG-20 laurate, PEG-32
laurate, PEG-32 dilaurate, PEG-12 oleate, PEG-15 oleate, PEG-20
oleate, PEG-20 dioleate, PEG-32 oleate, PEG-200 oleate, PEG-400
oleate, PEG-15 stearate, PEG-32 distearate, PEG-40 stearate,
PEG-100 stearate, PEG-20 dilaurate, PEG-25 glyceryl trioleate,
PEG-32 dioleate, PEG-20 glyceryl laurate, PEG-30 glyceryl laurate,
PEG-20 glyceryl stearate, PEG-20 glyceryl oleate, PEG-30 glyceryl
oleate, PEG-30 glyceryl laurate, PEG-40 glyceryl laurate, PEG-40
palm kernel oil, PEG-50 hydrogenated castor oil, PEG-40 castor oil,
PEG-35 castor oil, PEG-60 castor oil, PEG-40 hydrogenated castor
oil, PEG-60 hydrogenated castor oil, PEG-60 corn oil, PEG-6
caprate/caprylate glycerides, PEG-8 caprate/caprylate glycerides,
polyglyceryl-10 laurate, PEG-30 cholesterol, PEG-25 phyto sterol,
PEG-30 soya sterol, PEG-20 trioleate, PEG-40 sorbitan oleate,
PEG-80 sorbitan laurate, polysorbate 20, polysorbate 80, POE-9
lauryl ether, POE-23 lauryl ether, POE-10 oleyl ether, POE-20 oleyl
ether, POE-20 stearyl ether, tocopheryl PEG-100 succinate, PEG-24
cholesterol, polyglyceryl-10oleate, Tween 40, Tween 60, sucrose
monostearate, sucrose monolaurate, sucrose monopalmitate, PEG
10-100 nonyl phenol series, PEG 15-100 octyl phenol series, and
poloxamers.
Suitable lipophilic surfactants include, by way of example only:
fatty alcohols; glycerol fatty acid esters; acetylated glycerol
fatty acid esters; lower alcohol fatty acids esters; propylene
glycol fatty acid esters; sorbitan fatty acid esters; polyethylene
glycol sorbitan fatty acid esters; sterols and sterol derivatives;
polyoxyethylated sterols and sterol derivatives; polyethylene
glycol alkyl ethers; sugar esters; sugar ethers; lactic acid
derivatives of mono- and di-glycerides; hydrophobic
transesterification products of a polyol with at least one member
of the group consisting of glycerides, vegetable oils, hydrogenated
vegetable oils, fatty acids and sterols; oil-soluble
vitamins/vitamin derivatives; and mixtures thereof. Within this
group, preferred lipophilic surfactants include glycerol fatty acid
esters, propylene glycol fatty acid esters, and mixtures thereof,
or are hydrophobic transesterification products of a polyol with at
least one member of the group consisting of vegetable oils,
hydrogenated vegetable oils, and triglycerides.
In one embodiment, the composition may include a solubilizer to
ensure good solubilization and/or dissolution of the compound of
the present invention and to minimize precipitation of the compound
of the present invention. This can be especially important for
compositions for non-oral use, e.g., compositions for injection. A
solubilizer may also be added to increase the solubility of the
hydrophilic drug and/or other components, such as surfactants, or
to maintain the composition as a stable or homogeneous solution or
dispersion.
Examples of suitable solubilizers include, but are not limited to,
the following: alcohols and polyols, such as ethanol, isopropanol,
butanol, benzyl alcohol, ethylene glycol, propylene glycol,
butanediols and isomers thereof, glycerol, pentaerythritol,
sorbitol, mannitol, transcutol, dimethyl isosorbide, polyethylene
glycol, polypropylene glycol, polyvinylalcohol, hydroxypropyl
methylcellulose and other cellulose derivatives, cyclodextrins and
cyclodextrin derivatives; ethers of polyethylene glycols having an
average molecular weight of about 200 to about 6000, such as
tetrahydrofurfuryl alcohol PEG ether (glycofurol) or methoxy PEG;
amides and other nitrogen-containing compounds such as
2-pyrrolidone, 2-piperidone, .epsilon.-caprolactam,
N-alkylpyrrolidone, N-hydroxyalkylpyrrolidone, N-alkylpiperidone,
N-alkylcaprolactam, dimethylacetamide and polyvinylpyrrolidone;
esters such as ethyl propionate, tributylcitrate, acetyl
triethylcitrate, acetyl tributyl citrate, triethylcitrate, ethyl
oleate, ethyl caprylate, ethyl butyrate, triacetin, propylene
glycol monoacetate, propylene glycol diacetate,
.epsilon.-caprolactone and isomers thereof, .delta.-valerolactone
and isomers thereof, .beta.-butyrolactone and isomers thereof; and
other solubilizers known in the art, such as dimethyl acetamide,
dimethyl isosorbide, N-methyl pyrrolidones, monooctanoin,
diethylene glycol monoethyl ether, and water.
Mixtures of solubilizers may also be used. Examples include, but
not limited to, triacetin, triethylcitrate, ethyl oleate, ethyl
caprylate, dimethylacetamide, N-methylpyrrolidone,
N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropyl
methylcellulose, hydroxypropyl cyclodextrins, ethanol, polyethylene
glycol 200-100, glycofurol, transcutol, propylene glycol, and
dimethyl isosorbide. Particularly preferred solubilizers include
sorbitol, glycerol, triacetin, ethyl alcohol, PEG-400, glycofurol
and propylene glycol.
The amount of solubilizer that can be included is not particularly
limited. The amount of a given solubilizer may be limited to a
bioacceptable amount, which may be readily determined by one of
skill in the art. In some circumstances, it may be advantageous to
include amounts of solubilizers far in excess of bioacceptable
amounts, for example to maximize the concentration of the drug,
with excess solubilizer removed prior to providing the composition
to a patient using conventional techniques, such as distillation or
evaporation. Thus, if present, the solubilizer can be in a weight
ratio of 10%, 25%, 50%, 100%, or up to about 200% by weight, based
on the combined weight of the drug, and other excipients. If
desired, very small amounts of solubilizer may also be used, such
as 5%, 2%, 1% or even less. Typically, the solubilizer may be
present in an amount of about 1% to about 100%, more typically
about 5% to about 25% by weight.
The composition can further include one or more pharmaceutically
acceptable additives and excipients. Such additives and excipients
include, without limitation, detackifiers, anti-foaming agents,
buffering agents, polymers, antioxidants, preservatives, chelating
agents, viscomodulators, tonicifiers, flavorants, colorants,
odorants, opacifiers, suspending agents, binders, fillers,
plasticizers, lubricants, and mixtures thereof.
In addition, an acid or a base may be incorporated into the
composition to facilitate processing, to enhance stability, or for
other reasons. Examples of pharmaceutically acceptable bases
include amino acids, amino acid esters, ammonium hydroxide,
potassium hydroxide, sodium hydroxide, sodium hydrogen carbonate,
aluminum hydroxide, calcium carbonate, magnesium hydroxide,
magnesium aluminum silicate, synthetic aluminum silicate, synthetic
hydrocalcite, magnesium aluminum hydroxide, diisopropylethylamine,
ethanolamine, ethylenediamine, triethanolamine, triethylamine,
triisopropanolamine, trimethylamine,
tris(hydroxymethyl)aminomethane (TRIS) and the like. Also suitable
are bases that are salts of a pharmaceutically acceptable acid,
such as acetic acid, acrylic acid, adipic acid, alginic acid,
alkanesulfonic acid, amino acids, ascorbic acid, benzoic acid,
boric acid, butyric acid, carbonic acid, citric acid, fatty acids,
formic acid, fumaric acid, gluconic acid, hydroquinosulfonic acid,
isoascorbic acid, lactic acid, maleic acid, oxalic acid,
para-bromophenylsulfonic acid, propionic acid, p-toluenesulfonic
acid, salicylic acid, stearic acid, succinic acid, tannic acid,
tartaric acid, thioglycolic acid, toluenesulfonic acid, uric acid,
and the like. Salts of polyprotic acids, such as sodium phosphate,
disodium hydrogen phosphate, and sodium dihydrogen phosphate can
also be used. When the base is a salt, the cation can be any
convenient and pharmaceutically acceptable cation, such as
ammonium, alkali metals, alkaline earth metals, and the like.
Example may include, but not limited to, sodium, potassium,
lithium, magnesium, calcium and ammonium.
Suitable acids are pharmaceutically acceptable organic or inorganic
acids. Examples of suitable inorganic acids include hydrochloric
acid, hydrobromic acid, hydriodic acid, sulfuric acid, nitric acid,
boric acid, phosphoric acid, and the like. Examples of suitable
organic acids include acetic acid, acrylic acid, adipic acid,
alginic acid, alkanesulfonic acids, amino acids, ascorbic acid,
benzoic acid, boric acid, butyric acid, carbonic acid, citric acid,
fatty acids, formic acid, fumaric acid, gluconic acid,
hydroquinosulfonic acid, isoascorbic acid, lactic acid, maleic
acid, methanesulfonic acid, oxalic acid, para-bromophenylsulfonic
acid, propionic acid, p-toluenesulfonic acid, salicylic acid,
stearic acid, succinic acid, tannic acid, tartaric acid,
thioglycolic acid, toluenesulfonic acid, uric acid and the
like.
Pharmaceutical Compositions for Injection.
In some embodiments, the invention provides a pharmaceutical
composition for injection containing a compound of the present
invention and a pharmaceutical excipient suitable for injection.
Components and amounts of agents in the compositions are as
described herein.
The forms in which the novel compositions of the present invention
may be incorporated for administration by injection include aqueous
or oil suspensions, or emulsions, with sesame oil, corn oil,
cottonseed oil, or peanut oil, as well as elixirs, mannitol,
dextrose, or a sterile aqueous solution, and similar pharmaceutical
vehicles.
Aqueous solutions in saline are also conventionally used for
injection. Ethanol, glycerol, propylene glycol, liquid polyethylene
glycol, and the like (and suitable mixtures thereof), cyclodextrin
derivatives, and vegetable oils may also be employed. The proper
fluidity can be maintained, for example, by the use of a coating,
such as lecithin, for the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. The
prevention of the action of microorganisms can be brought about by
various antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
Sterile injectable solutions are prepared by incorporating the
compound of the present invention in the required amount in the
appropriate solvent with various other ingredients as enumerated
above, as required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, certain desirable
methods of preparation are vacuum-drying and freeze-drying
techniques which yield a powder of the active ingredient plus any
additional desired ingredient from a previously sterile-filtered
solution thereof.
Pharmaceutical Compositions for Topical (e.g., Transdermal)
Delivery.
In some embodiments, the invention provides a pharmaceutical
composition for transdermal delivery containing a compound of the
present invention and a pharmaceutical excipient suitable for
transdermal delivery.
Compositions of the present invention can be formulated into
preparations in solid, semi-solid, or liquid forms suitable for
local or topical administration, such as gels, water soluble
jellies, creams, lotions, suspensions, foams, powders, slurries,
ointments, solutions, oils, pastes, suppositories, sprays,
emulsions, saline solutions, dimethylsulfoxide (DMSO)-based
solutions. In general, carriers with higher densities are capable
of providing an area with a prolonged exposure to the active
ingredients. In contrast, a solution formulation may provide more
immediate exposure of the active ingredient to the chosen area.
The pharmaceutical compositions also may comprise suitable solid or
gel phase carriers or excipients, which are compounds that allow
increased penetration of, or assist in the delivery of, therapeutic
molecules across the stratum corneum permeability barrier of the
skin. There are many of these penetration-enhancing molecules known
to those trained in the art of topical formulation. Examples of
such carriers and excipients include, but are not limited to,
humectants (e.g., urea), glycols (e.g., propylene glycol), alcohols
(e.g., ethanol), fatty acids (e.g., oleic acid), surfactants (e.g.,
isopropyl myristate and sodium lauryl sulfate), pyrrolidones,
glycerol monolaurate, sulfoxides, terpenes (e.g., menthol), amines,
amides, alkanes, alkanols, water, calcium carbonate, calcium
phosphate, various sugars, starches, cellulose derivatives,
gelatin, and polymers such as polyethylene glycols.
Another exemplary formulation for use in the methods of the present
invention employs transdermal delivery devices ("patches"). Such
transdermal patches may be used to provide continuous or
discontinuous infusion of a compound of the present invention in
controlled amounts, either with or without another agent.
The construction and use of transdermal patches for the delivery of
pharmaceutical agents is well known in the art. See, e.g., U.S.
Pat. Nos. 5,023,252, 4,992,445 and 5,001,139. Such patches may be
constructed for continuous, pulsatile, or on demand delivery of
pharmaceutical agents.
Pharmaceutical Compositions for Inhalation.
Compositions for inhalation or insufflation include solutions and
suspensions in pharmaceutically acceptable, aqueous or organic
solvents, or mixtures thereof, and powders. The liquid or solid
compositions may contain suitable pharmaceutically acceptable
excipients as described supra. Preferably the compositions are
administered by the oral or nasal respiratory route for local or
systemic effect. Compositions in preferably pharmaceutically
acceptable solvents may be nebulized by use of inert gases.
Nebulized solutions may be inhaled directly from the nebulizing
device or the nebulizing device may be attached to a face mask
tent, or intermittent positive pressure breathing machine.
Solution, suspension, or powder compositions may be administered,
preferably orally or nasally, from devices that deliver the
formulation in an appropriate manner.
Other Pharmaceutical Compositions.
Pharmaceutical compositions may also be prepared from compositions
described herein and one or more pharmaceutically acceptable
excipients suitable for sublingual, buccal, rectal, intraosseous,
intraocular, intranasal, epidural, or intraspinal administration.
Preparations for such pharmaceutical compositions are well-known in
the art. See, e.g., See, e.g., Anderson, Philip O.; Knoben, James
E.; Troutman, William G, eds., Handbook of Clinical Drug Data,
Tenth Edition, McGraw-Hill, 2002; Pratt and Taylor, eds.,
Principles of Drug Action, Third Edition, Churchill Livingston, N.
Y., 1990; Katzung, ed., Basic and Clinical Pharmacology, Ninth
Edition, McGraw Hill, 20037ybg; Goodman and Gilman, eds., The
Pharmacological Basis of Therapeutics, Tenth Edition, McGraw Hill,
2001; Remingtons Pharmaceutical Sciences, 20th Ed., Lippincott
Williams & Wilkins, 2000; Martindale, The Extra Pharmacopoeia,
Thirty-Second Edition (The Pharmaceutical Press, London, 1999); all
of which are incorporated by reference herein in their
entirety.
Administration of the compounds or pharmaceutical composition of
the present invention can be effected by any method that enables
delivery of the compounds to the site of action. These methods
include oral routes, intraduodenal routes, parenteral injection
(including intravenous, intraarterial, subcutaneous, intramuscular,
intravascular, intraperitoneal or infusion), topical (e.g.
transdermal application), rectal administration, via local delivery
by catheter or stent or through inhalation. Compounds can also be
administered intraadiposally or intrathecally.
The amount of the compound administered will be dependent on the
mammal being treated, the severity of the disorder or condition,
the rate of administration, the disposition of the compound and the
discretion of the prescribing physician. However, an effective
dosage is in the range of about 0.001 to about 100 mg per kg body
weight per day, preferably about 1 to about 35 mg/kg/day, in single
or divided doses. For a 70 kg human, this would amount to about
0.05 to 7 g/day, preferably about 0.05 to about 2.5 g/day. In some
instances, dosage levels below the lower limit of the aforesaid
range may be more than adequate, while in other cases still larger
doses may be employed without causing any harmful side effect, e.g.
by dividing such larger doses into several small doses for
administration throughout the day.
In some embodiments, a compound of the invention is administered in
a single dose. Typically, such administration will be by injection,
e.g., intravenous injection, in order to introduce the agent
quickly. However, other routes may be used as appropriate. A single
dose of a compound of the invention may also be used for treatment
of an acute condition.
In some embodiments, a compound of the invention is administered in
multiple doses. Dosing may be about once, twice, three times, four
times, five times, six times, or more than six times per day.
Dosing may be about once a month, once every two weeks, once a
week, or once every other day. In another embodiment a compound of
the invention and another agent are administered together about
once per day to about 6 times per day. In another embodiment the
administration of a compound of the invention and an agent
continues for less than about 7 days. In yet another embodiment the
administration continues for more than about 6, 10, 14, 28 days,
two months, six months, or one year. In some cases, continuous
dosing is achieved and maintained as long as necessary.
Administration of the agents of the invention may continue as long
as necessary. In some embodiments, an agent of the invention is
administered for more than 1, 2, 3, 4, 5, 6, 7, 14, or 28 days. In
some embodiments, an agent of the invention is administered for
less than 28, 14, 7, 6, 5, 4, 3, 2, or 1 day. In some embodiments,
an agent of the invention is administered chronically on an ongoing
basis, e.g., for the treatment of chronic effects.
An effective amount of a compound of the invention may be
administered in either single or multiple doses by any of the
accepted modes of administration of agents having similar
utilities, including rectal, buccal, intranasal and transdermal
routes, by intra-arterial injection, intravenously,
intraperitoneally, parenterally, intramuscularly, subcutaneously,
orally, topically, or as an inhalant.
The compositions of the invention may also be delivered via an
impregnated or coated device such as a stent, for example, or an
artery-inserted cylindrical polymer. Such a method of
administration may, for example, aid in the prevention or
amelioration of restenosis following procedures such as balloon
angioplasty. Without being bound by theory, compounds of the
invention may slow or inhibit the migration and proliferation of
smooth muscle cells in the arterial wall which contribute to
restenosis. A compound of the invention may be administered, for
example, by local delivery from the struts of a stent, from a stent
graft, from grafts, or from the cover or sheath of a stent. In some
embodiments, a compound of the invention is admixed with a matrix.
Such a matrix may be a polymeric matrix, and may serve to bond the
compound to the stent. Polymeric matrices suitable for such use,
include, for example, lactone-based polyesters or copolyesters such
as polylactide, polycaprolactonglycolide, polyorthoesters,
polyanhydrides, polyaminoacids, polysaccharides, polyphosphazenes,
poly(ether-ester) copolymers (e.g. PEO-PLLA); polydimethylsiloxane,
poly(ethylene-vinylacetate), acrylate-based polymers or copolymers
(e.g. polyhydroxyethyl methylmethacrylate, polyvinyl
pyrrolidinone), fluorinated polymers such as
polytetrafluoroethylene and cellulose esters. Suitable matrices may
be nondegrading or may degrade with time, releasing the compound or
compounds. Compounds of the invention may be applied to the surface
of the stent by various methods such as dip/spin coating, spray
coating, dip-coating, and/or brush-coating. The compounds may be
applied in a solvent and the solvent may be allowed to evaporate,
thus forming a layer of compound onto the stent. Alternatively, the
compound may be located in the body of the stent or graft, for
example in microchannels or micropores. When implanted, the
compound diffuses out of the body of the stent to contact the
arterial wall. Such stents may be prepared by dipping a stent
manufactured to contain such micropores or microchannels into a
solution of the compound of the invention in a suitable solvent,
followed by evaporation of the solvent. Excess drug on the surface
of the stent may be removed via an additional brief solvent wash.
In yet other embodiments, compounds of the invention may be
covalently linked to a stent or graft. A covalent linker may be
used which degrades in vivo, leading to the release of the compound
of the invention. Any bio-labile linkage may be used for such a
purpose, such as ester, amide or anhydride linkages. Compounds of
the invention may additionally be administered intravascularly from
a balloon used during angioplasty. Extravascular administration of
the compounds via the pericard or via advential application of
formulations of the invention may also be performed to decrease
restenosis.
A variety of stent devices which may be used as described are
disclosed, for example, in the following references, all of which
are hereby incorporated by reference: U.S. Pat. No. 5,451,233; U.S.
Pat. No. 5,040,548; U.S. Pat. No. 5,061,273; U.S. Pat. No.
5,496,346; U.S. Pat. No. 5,292,331; U.S. Pat. No. 5,674,278; U.S.
Pat. No. 3,657,744; U.S. Pat. No. 4,739,762; U.S. Pat. No.
5,195,984; U.S. Pat. No. 5,292,331; U.S. Pat. No. 5,674,278; U.S.
Pat. No. 5,879,382; U.S. Pat. No. 6,344,053.
The compounds of the invention may be administered in dosages. It
is known in the art that due to intersubject variability in
compound pharmacokinetics, individualization of dosing regimen is
necessary for optimal therapy. Dosing for a compound of the
invention may be found by routine experimentation in light of the
instant disclosure.
When a compound of the invention, is administered in a composition
that comprises one or more agents, and the agent has a shorter
half-life than the compound of the invention unit dose forms of the
agent and the compound of the invention may be adjusted
accordingly.
The subject pharmaceutical composition may, for example, be in a
form suitable for oral administration as a tablet, capsule, pill,
powder, sustained release formulations, solution, suspension, for
parenteral injection as a sterile solution, suspension or emulsion,
for topical administration as an ointment or cream or for rectal
administration as a suppository. The pharmaceutical composition may
be in unit dosage forms suitable for single administration of
precise dosages. The pharmaceutical composition will include a
conventional pharmaceutical carrier or excipient and a compound
according to the invention as an active ingredient. In addition, it
may include other medicinal or pharmaceutical agents, carriers,
adjuvants, etc.
Exemplary parenteral administration forms include solutions or
suspensions of active compound in sterile aqueous solutions, for
example, aqueous propylene glycol or dextrose solutions. Such
dosage forms can be suitably buffered, if desired.
The activity of the compounds of the present invention may be
determined by the following procedure, as well as the procedure
described in the examples below. The activity of the kinase is
assessed by measuring the incorporation of
.gamma.-.sup.33P-phosphate from .gamma.-.sup.33P-ATP onto
N-terminal His tagged substrate, which is expressed in E. coli and
is purified by conventional methods, in the presence of the kinase.
The assay is carried out in 96-well polypropylene plate. The
incubation mixture (100, .mu.L) comprises of 25 mM Hepes, pH 7.4,
10 mM MgCl.sub.2, 5 mM .beta.-glycerolphosphate, 100 .mu.M
Na-orthovanadate, 5 mM DTT, 5 nM kinase, and 1 .mu.M substrate
Inhibitors are suspended in DMSO, and all reactions, including
controls are performed at a final concentration of 1% DMSO.
Reactions are initiated by the addition of 10 .mu.M ATP (with 0.5
.mu.Ci .gamma.-.sup.33P-ATP/well) and incubated at ambient
temperature for 45 minutes. Equal volume of 25% TCA is added to
stop the reaction and precipitate the proteins. Precipitated
proteins are trapped onto glass fiber B filterplates, and excess
labeled ATP washed off using a Tomtec MACH III harvestor. Plates
are allowed to air-dry prior to adding 30 .mu.L/well of Packard
Microscint 20, and plates are counted using a Packard TopCount.
The invention also provides kits. The kits include a compound or
compounds of the present invention as described herein, in suitable
packaging, and written material that can include instructions for
use, discussion of clinical studies, listing of side effects, and
the like. Such kits may also include information, such as
scientific literature references, package insert materials,
clinical trial results, and/or summaries of these and the like,
which indicate or establish the activities and/or advantages of the
composition, and/or which describe dosing, administration, side
effects, drug interactions, or other information useful to the
health care provider. Such information may be based on the results
of various studies, for example, studies using experimental animals
involving in vivo models and studies based on human clinical
trials. The kit may further contain another agent. In some
embodiments, the compound of the present invention and the agent
are provided as separate compositions in separate containers within
the kit. In some embodiments, the compound of the present invention
and the agent are provided as a single composition within a
container in the kit. Suitable packaging and additional articles
for use (e.g., measuring cup for liquid preparations, foil wrapping
to minimize exposure to air, and the like) are known in the art and
may be included in the kit. Kits described herein can be provided,
marketed and/or promoted to health providers, including physicians,
nurses, pharmacists, formulary officials, and the like. Kits may
also, in some embodiments, be marketed directly to the
consumer.
Methods
The invention also provides methods of using the compounds or
pharmaceutical compositions of the present invention to treat
disease conditions, including but not limited to diseases
associated with malfunctioning of one or more types of PI3 kinase.
A detailed description of conditions and disorders mediated by
p110.delta. kinase activity is set forth in Sadu et al., WO
01/81346, which is incorporated herein by reference in its entirety
for all purposes.
The treatment methods provided herein comprise administering to the
subject a therapeutically effective amount of a compound of the
invention. In one embodiment, the present invention provides a
method of treating an inflammation disorder, including autoimmune
diseases in a mammal. The method comprises administering to said
mammal a therapeutically effective amount of a compound of the
present invention, or a pharmaceutically acceptable salt, ester,
prodrug, solvate, hydrate or derivative thereof. Examples of
autoimmune diseases includes but is not limited to acute
disseminated encephalomyelitis (ADEM), Addison's disease,
antiphospholipid antibody syndrome (APS), aplastic anemia,
autoimmune hepatitis, coeliac disease, Crohn's disease, Diabetes
mellitus (type 1), Goodpasture's syndrome, Graves' disease,
Guillain-Barre syndrome (GBS), Hashimoto's disease, lupus
erythematosus, multiple sclerosis, myasthenia gravis, opsoclonus
myoclonus syndrome (OMS), optic neuritis, Ord's thyroiditis,
oemphigus, polyarthritis, primary biliary cirrhosis, psoriasis,
rheumatoid arthritis, Reiter's syndrome, Takayasu's arteritis,
temporal arteritis (also known as "giant cell arteritis"), warm
autoimmune hemolytic anemia, Wegener's granulomatosis, alopecia
universalis, Chagas' disease, chronic fatigue syndrome,
dysautonomia, endometriosis, hidradenitis suppurativa, interstitial
cystitis, neuromyotonia, sarcoidosis, scleroderma, ulcerative
colitis, vitiligo, and vulvodynia. Other disorders include
bone-resorption disorders and thromobsis.
In some embodiments, the method of treating inflammatory or
autoimmune diseases comprises administering to a subject (e.g. a
mammal) a therapeutically effective amount of one or more compounds
of the present invention that selectively inhibit PI3K-.delta.
and/or PI3K-.gamma. as compared to all other type I PI3 kinases.
Such selective inhibition of PI3K-.delta. and/or PI3K-.gamma. may
be advantageous for treating any of the diseases or conditions
described herein. For example, selective inhibition of PI3K-.delta.
may inhibit inflammatory responses associated with inflammatory
diseases, autoimmune disease, or diseases related to an undesirable
immune response including but not limited to asthma, emphysema,
allergy, dermatitis, rheumatoid arthritis, psoriasis, lupus
erythematosus, or graft versus host disease. Selective inhibition
of PI3K-.delta. may further provide for a reduction in the
inflammatory or undesirable immune response without a concomitant
reduction in the ability to reduce a bacterial, viral, and/or
fungal infection. Selective inhibition of both PI3K-.delta. and
PI3K-.gamma. may be advantageous for inhibiting the inflammatory
response in the subject to a greater degree than that would be
provided for by inhibitors that selectively inhibit PI3K-.delta. or
PI3K-.gamma. alone. In one aspect, one or more of the subject
methods are effective in reducing antigen specific antibody
production in vivo by about 2-fold, 3-fold, 4-fold, 5-fold,
7.5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 250-fold, 500-fold,
750-fold, or about 1000-fold or more. In another aspect, one or
more of the subject methods are effective in reducing antigen
specific IgG3 and/or IgGM production in vivo by about 2-fold,
3-fold, 4-fold, 5-fold, 7.5-fold, 10-fold, 25-fold, 50-fold,
100-fold, 250-fold, 500-fold, 750-fold, or about 1000-fold or
more.
In one aspect, one of more of the subject methods are effective in
ameliorating symptoms associated with rheumatoid arthritis
including but not limited to a reduction in the swelling of joints,
a reduction in serum anti-collagen levels, and/or a reduction in
joint pathology such as bone resorption, cartilage damage, pannus,
and/or inflammation. In another aspect, the subject methods are
effective in reducing ankle inflammation by at least about 2%, 5%,
10%, 15%, 20%, 25%, 30%, 50%, 60%, or about 75% to 90%. In another
aspect, the subject methods are effective in reducing knee
inflammation by at least about 2%, 5%, 10%, 15%, 20%, 25%, 30%,
50%, 60%, or about 75% to 90% or more. In still another aspect, the
subject methods are effective in reducing serum anti-type II
collagen levels by at least about 10%, 12%, 15%, 20%, 24%, 25%,
30%, 35%, 50%, 60%, 75%, 80%, 86%, 87%, or about 90% or more. In
another aspect, the subject methods are effective in reducing ankle
histopathology scores by about 5%, 10%, 15%, 20%, 25%, 30%, 40%,
50%, 60%, 75%, 80%, 90% or more. In still another aspect, the
subject methods are effective in reducing knee histopathology
scores by about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 75%,
80%, 90% or more.
In other embodiments, the present invention provides methods of
using the compounds or pharmaceutical compositions to treat
respiratory diseases including but not limited to diseases
affecting the lobes of lung, pleural cavity, bronchial tubes,
trachea, upper respiratory tract, or the nerves and muscle for
breathing. For example, methods are provided to treat obstructive
pulmonary disease. Chronic obstructive pulmonary disease (COPD) is
an umbrella term for a group of respiratory tract diseases that are
characterized by airflow obstruction or limitation. Conditions
included in this umbrella term are: chronic bronchitis, emphysema,
and bronchiectasis.
In another embodiment, the compounds described herein are used for
the treatment of asthma. Also, the compounds or pharmaceutical
compositions described herein may be used for the treatment of
endotoxemia and sepsis. In one embodiment, the compounds or
pharmaceutical compositions described herein are used to for the
treatment of rheumatoid arthritis (RA). In yet another embodiment,
the compounds or pharmaceutical compositions described herein is
used for the treatment of contact or atopic dermatitis. Contact
dermatitis includes irritant dermatitis, phototoxic dermatitis,
allergic dermatitis, photoallergic dermatitis, contact urticaria,
systemic contact-type dermatitis and the like. Irritant dermatitis
can occur when too much of a substance is used on the skin of when
the skin is sensitive to certain substance. Atopic dermatitis,
sometimes called eczema, is a kind of dermatitis, an atopic skin
disease.
The invention also relates to a method of treating a
hyperproliferative disorder in a mammal that comprises
administering to said mammal a therapeutically effective amount of
a compound of the present invention, or a pharmaceutically
acceptable salt, ester, prodrug, solvate, hydrate or derivative
thereof. In some embodiments, said method relates to the treatment
of cancer such as acute myeloid leukemia, thymus, brain, lung,
squamous cell, skin, eye, retinoblastoma, intraocular melanoma,
oral cavity and oropharyngeal, bladder, gastric, stomach,
pancreatic, bladder, breast, cervical, head, neck, renal, kidney,
liver, ovarian, prostate, colorectal, esophageal, testicular,
gynecological, thyroid, CNS, PNS, AIDS-related (e.g. Lymphoma and
Kaposi's Sarcoma) or viral-induced cancer. In some embodiments,
said method relates to the treatment of a non-cancerous
hyperproliferative disorder such as benign hyperplasia of the skin
(e. g., psoriasis), restenosis, or prostate (e. g., benign
prostatic hypertrophy (BPH)).
The invention also relates to a method of treating diseases related
to vasculogenesis or angiogenesis in a mammal that comprises
administering to said mammal a therapeutically effective amount of
a compound of the present invention, or a pharmaceutically
acceptable salt, ester, prodrug, solvate, hydrate or derivative
thereof. In some embodiments, said method is for treating a disease
selected from the group consisting of tumor angiogenesis, chronic
inflammatory disease such as rheumatoid arthritis, atherosclerosis,
inflammatory bowel disease, skin diseases such as psoriasis,
eczema, and scleroderma, diabetes, diabetic retinopathy,
retinopathy of prematurity, age-related macular degeneration,
hemangioma, glioma, melanoma, Kaposi's sarcoma and ovarian, breast,
lung, pancreatic, prostate, colon and epidermoid cancer.
Patients that can be treated with compounds of the present
invention, or pharmaceutically acceptable salt, ester, prodrug,
solvate, hydrate or derivative of said compounds, according to the
methods of this invention include, for example, patients that have
been diagnosed as having psoriasis; restenosis; atherosclerosis;
BPH; breast cancer such as a ductal carcinoma in duct tissue in a
mammary gland, medullary carcinomas, colloid carcinomas, tubular
carcinomas, and inflammatory breast cancer; ovarian cancer,
including epithelial ovarian tumors such as adenocarcinoma in the
ovary and an adenocarcinoma that has migrated from the ovary into
the abdominal cavity; uterine cancer; cervical cancer such as
adenocarcinoma in the cervix epithelial including squamous cell
carcinoma and adenocarcinomas; prostate cancer, such as a prostate
cancer selected from the following: an adenocarcinoma or an
adenocarcinoma that has migrated to the bone; pancreatic cancer
such as epitheliod carcinoma in the pancreatic duct tissue and an
adenocarcinoma in a pancreatic duct; bladder cancer such as a
transitional cell carcinoma in urinary bladder, urothelial
carcinomas (transitional cell carcinomas), tumors in the urothelial
cells that line the bladder, squamous cell carcinomas,
adenocarcinomas, and small cell cancers; leukemia such as acute
myeloid leukemia (AML), acute lymphocytic leukemia, chronic
lymphocytic leukemia, chronic myeloid leukemia, hairy cell
leukemia, myelodysplasia, myeloproliferative disorders, acute
myelogenous leukemia (AML), chronic myelogenous leukemia (CML),
mastocytosis, chronic lymphocytic leukemia (CLL), multiple myeloma
(MM), and myelodysplastic syndrome (MDS); bone cancer; lung cancer
such as non-small cell lung cancer (NSCLC), which is divided into
squamous cell carcinomas, adenocarcinomas, and large cell
undifferentiated carcinomas, and small cell lung cancer; skin
cancer such as basal cell carcinoma, melanoma, squamous cell
carcinoma and actinic keratosis, which is a skin condition that
sometimes develops into squamous cell carcinoma; eye
retinoblastoma; cutaneous or intraocular (eye) melanoma; primary
liver cancer (cancer that begins in the liver); kidney cancer;
thyroid cancer such as papillary, follicular, medullary and
anaplastic; AIDS-related lymphoma such as diffuse large B-cell
lymphoma, B-cell immunoblastic lymphoma and small non-cleaved cell
lymphoma; Kaposi's Sarcoma; viral-induced cancers including
hepatitis B virus (HBV), hepatitis C virus (HCV), and
hepatocellular carcinoma; human lymphotropic virus-type 1 (HTLV-1)
and adult T-cell leukemia/lymphoma; and human papilloma virus (HPV)
and cervical cancer; central nervous system cancers (CNS) such as
primary brain tumor, which includes gliomas (astrocytoma,
anaplastic astrocytoma, or glioblastoma multiforme),
Oligodendroglioma, Ependymoma, Meningioma, Lymphoma, Schwannoma,
and Medulloblastoma; peripheral nervous system (PNS) cancers such
as acoustic neuromas and malignant peripheral nerve sheath tumor
(MPNST) including neurofibromas and schwannomas, malignant fibrous
cytoma, malignant fibrous histiocytoma, malignant meningioma,
malignant mesothelioma, and malignant mixed Mullerian tumor; oral
cavity and oropharyngeal cancer such as, hypopharyngeal cancer,
laryngeal cancer, nasopharyngeal cancer, and oropharyngeal cancer;
stomach cancer such as lymphomas, gastric stromal tumors, and
carcinoid tumors; testicular cancer such as germ cell tumors
(GCTs), which include seminomas and nonseminomas, and gonadal
stromal tumors, which include Leydig cell tumors and Sertoli cell
tumors; thymus cancer such as to thymomas, thymic carcinomas,
Hodgkin disease, non-Hodgkin lymphomas carcinoids or carcinoid
tumors; rectal cancer; and colon cancer.
The invention also relates to a method of treating diabetes in a
mammal that comprises administering to said mammal a
therapeutically effective amount of a compound of the present
invention, or a pharmaceutically acceptable salt, ester, prodrug,
solvate, hydrate or derivative thereof.
In addition, the compounds described herein may be used to treat
acne.
In addition, the compounds described herein may be used for the
treatment of arteriosclerosis, including atherosclerosis.
Arteriosclerosis a general term describing any hardening of medium
or large arteries. Atherosclerosis a hardening of an artery
specifically due to an atheromatous plaque.
Further the compounds described herein may be used for the
treatment of glomerulonephritis. Glomerulonephritis a primary or
secondary autoimmune renal disease characterized by inflammation of
the glomeruli. It may be asymptomatic, or present with hematuria
and/or proteinuria. There are many recognized types, divided in
acute, subacute or chronic glomerulonephritis. Causes are
infectious (bacterial, viral or parasitic pathogens), autoimmune or
paraneoplastic.
Additionally, the compounds described herein may be used for the
treatment of bursitis, lupus, acute disseminated encephalomyelitis
(ADEM), addison's disease, antiphospholipid antibody syndrome
(APS), aplastic anemia, autoimmune hepatitis, coeliac disease,
crohn's disease, diabetes mellitus (type 1), goodpasture's
syndrome, graves' disease, guillain-barre syndrome (GBS),
hashimoto's disease, inflammatory bowel disease, lupus
erythematosus, myasthenia gravis, opsoclonus myoclonus syndrome
(OMS), optic neuritis, ord's thyroiditis, ostheoarthritis,
uveoretinitis, pemphigus, polyarthritis, primary biliary cirrhosis,
reiter's syndrome, takayasu's arteritis, temporal arteritis, warm
autoimmune hemolytic anemia, wegener's granulomatosis, alopecia
universalis, chagas' disease, chronic fatigue syndrome,
dysautonomia, endometriosis, hidradenitis suppurativa, interstitial
cystitis, neuromyotonia, sarcoidosis, scleroderma, ulcerative
colitis, vitiligo, vulvodynia, appendicitis, arteritis, arthritis,
blepharitis, bronchiolitis, bronchitis, cervicitis, cholangitis,
cholecystitis, chorioamnionitis, colitis, conjunctivitis, cystitis,
dacryoadenitis, dermatomyositis, endocarditis, endometritis,
enteritis, enterocolitis, epicondylitis, epididymitis, fasciitis,
fibrositis, gastritis, gastroenteritis, gingivitis, hepatitis,
hidradenitis, ileitis, iritis, laryngitis, mastitis, meningitis,
myelitis, myocarditis, myositis, nephritis, omphalitis, oophoritis,
orchitis, osteitis, otitis, pancreatitis, parotitis, pericarditis,
peritonitis, pharyngitis, pleuritis, phlebitis, pneumonitis,
proctitis, prostatitis, pyelonephritis, rhinitis, salpingitis,
sinusitis, stomatitis, synovitis, tendonitis, tonsillitis, uveitis,
vaginitis, vasculitis, or vulvitis.
The invention also relates to a method of treating a cardiovascular
disease in a mammal that comprises administering to said mammal a
therapeutically effective amount of a compound of the present
invention, or a pharmaceutically acceptable salt, ester, prodrug,
solvate, hydrate or derivative thereof. Examples of cardiovascular
conditions include, but are not limited to, atherosclerosis,
restenosis, vascular occlusion and carotid obstructive disease.
In another aspect, the present invention provides methods of
disrupting the function of a leukocyte or disrupting a function of
an osteoclast. The method includes contacting the leukocyte or the
osteoclast with a function disrupting amount of a compound of the
invention.
In another aspect of the present invention, methods are provided
for treating ophthalmic disease by administering one or more of the
subject compounds or pharmaceutical compositions to the eye of a
subject.
Methods are further provided for administering the compounds of the
present invention via eye drop, intraocular injection, intravitreal
injection, topically, or through the use of a drug eluting device,
microcapsule, implant, or microfluidic device. In some cases, the
compounds of the present invention are administered with a carrier
or excipient that increases the intraocular penetrance of the
compound such as an oil and water emulsion with colloid particles
having an oily core surrounded by an interfacial film.
In some cases, the colloid particles include at least one cationic
agent and at least one non-ionic surfactant such as a poloxamer,
tyloxapol, a polysorbate, a polyoxyethylene castor oil derivative,
a sorbitan ester, or a polyoxyl stearate. In some cases, the
cationic agent is an alkylamine, a tertiary alkyl amine, a
quarternary ammonium compound, a cationic lipid, an amino alcohol,
a biguanidine salt, a cationic compound or a mixture thereof. In
some cases the cationic agent is a biguanidine salt such as
chlorhexidine, polyaminopropyl biguanidine, phenformin,
alkylbiguanidine, or a mixture thereof. In some cases, the
quaternary ammonium compound is a benzalkonium halide, lauralkonium
halide, cetrimide, hexadecyltrimethylammonium halide,
tetradecyltrimethylammonium halide, dodecyltrimethylammonium
halide, cetrimonium halide, benzethonium halide, behenalkonium
halide, cetalkonium halide, cetethyldimonium halide,
cetylpyridinium halide, benzododecinium halide, chlorallyl
methenamine halide, rnyristylalkonium halide, stearalkonium halide
or a mixture of two or more thereof. In some cases, cationic agent
is a benzalkonium chloride, lauralkonium chloride, benzododecinium
bromide, benzethenium chloride, hexadecyltrimethylammonium bromide,
tetradecyltrimethylammonium bromide, dodecyltrimethylammonium
bromide or a mixture of two or more thereof. In some cases, the oil
phase is mineral oil and light mineral oil, medium chain
triglycerides (MCT), coconut oil; hydrogenated oils comprising
hydrogenated cottonseed oil, hydrogenated palm oil, hydrogenate
castor oil or hydrogenated soybean oil; polyoxyethylene
hydrogenated castor oil derivatives comprising poluoxyl-40
hydrogenated castor oil, polyoxyl-60 hydrogenated castor oil or
polyoxyl-100 hydrogenated castor oil.
The invention further provides methods of modulating kinase
activity by contacting a kinase with an amount of a compound of the
invention sufficient to modulate the activity of the kinase.
Modulate can be inhibiting or activating kinase activity. In some
embodiments, the invention provides methods of inhibiting kinase
activity by contacting a kinase with an amount of a compound of the
invention sufficient to inhibit the activity of the kinase. In some
embodiments, the invention provides methods of inhibiting kinase
activity in a solution by contacting said solution with an amount
of a compound of the invention sufficient to inhibit the activity
of the kinase in said solution. In some embodiments, the invention
provides methods of inhibiting kinase activity in a cell by
contacting said cell with an amount of a compound of the invention
sufficient to inhibit the activity of the kinase in said cell. In
some embodiments, the invention provides methods of inhibiting
kinase activity in a tissue by contacting said tissue with an
amount of a compound of the invention sufficient to inhibit the
activity of the kinase in said tissue. In some embodiments, the
invention provides methods of inhibiting kinase activity in an
organism by contacting said organism with an amount of a compound
of the invention sufficient to inhibit the activity of the kinase
in said organism. In some embodiments, the invention provides
methods of inhibiting kinase activity in an animal by contacting
said animal with an amount of a compound of the invention
sufficient to inhibit the activity of the kinase in said animal. In
some embodiments, the invention provides methods of inhibiting
kinase activity in a mammal by contacting said mammal with an
amount of a compound of the invention sufficient to inhibit the
activity of the kinase in said mammal. In some embodiments, the
invention provides methods of inhibiting kinase activity in a human
by contacting said human with an amount of a compound of the
invention sufficient to inhibit the activity of the kinase in said
human. In some embodiments, the % of kinase activity after
contacting a kinase with a compound of the invention is less than
1, 5, 10, 20, 30, 40, 50, 60, 70, 80 90, 95, or 99% of the kinase
activity in the absence of said contacting step.
In some embodiments, the kinase is a lipid kinase or a protein
kinase. In some embodiments, the kinase is selected from the group
consisting of PI3 kinase including different isorforms such as PI3
kinase .alpha., PI3 kinase .beta., PI3 kinase .gamma., PI3 kinase
.delta.; DNA-PK; mTor; Abl, VEGFR, Ephrin receptor B4 (EphB4); TEK
receptor tyrosine kinase (TIE2); FMS-related tyrosine kinase 3
(FLT-3); Platelet derived growth factor receptor (PDGFR); RET; ATM;
ATR; hSmg-1; Hck; Src; Epidermal growth factor receptor (EGFR);
KIT; Inulsin Receptor (IR) and IGFR.
The invention further provides methods of modulating PI3 kinase
activity by contacting a PI3 kinase with an amount of a compound of
the invention sufficient to modulate the activity of the PI3
kinase. Modulate can be inhibiting or activating PI3 kinase
activity. In some embodiments, the invention provides methods of
inhibiting PI3 kinase activity by contacting a PI3 kinase with an
amount of a compound of the invention sufficient to inhibit the
activity of the PI3 kinase. In some embodiments, the invention
provides methods of inhibiting PI3 kinase activity. Such inhibition
can take place in solution, in a cell expressing one or more PI3
kinases, in a tissue comprising a cell expressing one or more PI3
kinases, or in an organism expressing one or more PI3 kinases. In
some embodiments, the invention provides methods of inhibiting PI3
kinase activity in an animal (including mammal such as humans) by
contacting said animal with an amount of a compound of the
invention sufficient to inhibit the activity of the PI3 kinase in
said animal.
Combination Treatment
The present invention also provides methods for combination
therapies in which an agent known to modulate other pathways, or
other components of the same pathway, or even overlapping sets of
target enzymes are used in combination with a compound of the
present invention, or a pharmaceutically acceptable salt, ester,
prodrug, solvate, hydrate or derivative thereof. In one aspect,
such therapy includes but is not limited to the combination of the
subject compound with chemotherapeutic agents, therapeutic
antibodies, and radiation treatment, to provide a synergistic or
additive therapeutic effect.
In one aspect, the compounds or pharmaceutical compositions of the
present invention may present synergistic or additive efficacy when
administered in combination with agents that inhibit IgE production
or activity. Such combination can reduce the undesired effect of
high level of IgE associated with the use of one or more
PI3K.delta. inhibitors, if such effect occurs. This may be
particularly useful in treatment of autoimmune and inflammatory
disorders (AIID) such as rheumatoid arthritis. Additionally, the
administration of PI3K.delta. or PI3K.delta./.gamma. inhibitors of
the present invention in combination with inhibitors of mTOR may
also exhibit synergy through enhanced inhibition of the PI3K
pathway.
In a separate but related aspect, the present invention provides a
combination treatment of a disease associated with PI3K.delta.
comprising administering to a PI3K.delta. inhibitor and an agent
that inhibits IgE production or activity. Other exemplary
PI3K.delta. inhibitors are applicable and they are described, e.g.,
U.S. Pat. No. 6,800,620. Such combination treatment is particularly
useful for treating autoimmune and inflammatory diseases (AIID)
including but not limited to rheumatoid arthritis.
Agents that inhibit IgE production are known in the art and they
include but are not limited to one or more of TEI-9874,
2-(4-(6-cyclohexyloxy-2-naphtyloxy)phenylacetamide)benzoic acid,
rapamycin, rapamycin analogs (i.e. rapalogs), TORC1 inhibitors,
TORC2 inhibitors, and any other compounds that inhibit mTORC1 and
mTORC2. Agents that inhibit IgE activity include, for example,
anti-IgE antibodies such as for example Omalizumab and TNX-901.
For treatment of autoimmune diseases, the subject compounds or
pharmaceutical compositions can be used in combination with
commonly prescribed drugs including but not limited to Enbrel.RTM.,
Remicade.RTM., Humira.RTM., Avonex.RTM., and Rebif.RTM.. For
treatment of respiratory diseases, the subject compounds or
pharmaceutical compositions can be administered in combination with
commonly prescribed drugs including but not limited to Xolair.RTM.,
Advair.RTM., Singulair.RTM., and Spiriva.RTM..
The compounds of the invention may be formulated or administered in
conjunction with other agents that act to relieve the symptoms of
inflammatory conditions such as encephalomyelitis, asthma, and the
other diseases described herein. These agents include non-steroidal
anti-inflammatory drugs (NSAIDs), e.g. acetylsalicylic acid;
ibuprofen; naproxen; indomethacin; nabumetone; tolmetin; etc.
Corticosteroids are used to reduce inflammation and suppress
activity of the immune system. The most commonly prescribed drug of
this type is Prednisone. Chloroquine (Aralen) or hydroxychloroquine
(Plaquenil) may also be very useful in some individuals with lupus.
They are most often prescribed for skin and joint symptoms of
lupus. Azathioprine (Imuran) and cyclophosphamide (Cytoxan)
suppress inflammation and tend to suppress the immune system. Other
agents, e.g. methotrexate and cyclosporin are used to control the
symptoms of lupus. Anticoagulants are employed to prevent blood
from clotting rapidly. They range from aspirin at very low dose
which prevents platelets from sticking, to heparin/coumadin.
In another one aspect, this invention also relates to a
pharmaceutical composition for inhibiting abnormal cell growth in a
mammal which comprises an amount of a compound of the present
invention, or a pharmaceutically acceptable salt, ester, prodrug,
solvate, hydrate or derivative thereof, in combination with an
amount of an anti-cancer agent (e.g. a chemotherapeutic agent).
Many chemotherapeutics are presently known in the art and can be
used in combination with the compounds of the invention.
In some embodiments, the chemotherapeutic is selected from the
group consisting of mitotic inhibitors, alkylating agents,
anti-metabolites, intercalating antibiotics, growth factor
inhibitors, cell cycle inhibitors, enzymes, topoisomerase
inhibitors, biological response modifiers, anti-hormones,
angiogenesis inhibitors, and anti-androgens. Non-limiting examples
are chemotherapeutic agents, cytotoxic agents, and non-peptide
small molecules such as Gleevec (Imatinib Mesylate), Velcade
(bortezomib), Casodex (bicalutamide), Iressa (gefitinib), and
Adriamycin as well as a host of chemotherapeutic agents.
Non-limiting examples of chemotherapeutic agents include alkylating
agents such as thiotepa and cyclosphosphamide (CYTOXAN.TM.); alkyl
sulfonates such as busulfan, improsulfan and piposulfan; aziridines
such as benzodopa, carboquone, meturedopa, and uredopa;
ethylenimines and methylamelamines including altretamine,
triethylenemelamine, trietylenephosphoramide,
triethylenethiophosphaoramide and trimethylolomelamine; nitrogen
mustards (i.e., compounds containing at least one
N--CH.sub.2--CH.sub.2--Cl moiety (see, e.g., Stedman's Medical
Dictionary 27.sup.th Ed. (2000), "nitrogen mustard" under
"mustard")) such as chlorambucil, chlornaphazine, cyclophosphamide,
estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide
hydrochloride, melphalan, novembichin, phenesterine, prednimustine,
trofosfamide, uracil mustard; nitrosureas such as carmustine,
chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;
antibiotics such as aclacinomysins, actinomycin, authramycin,
azaserine, bleomycins, cactinomycin, calicheamicin, carabicin,
carminomycin, carzinophilin, Casodex.TM., chromomycins,
dactinomycin, daunorubicin, detorubicin,
6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,
idarubicin, marcellomycin, mitomycins, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate and 5-fluorouracil (5-FU); folic acid analogues such
as denopterin, methotrexate, pteropterin, trimetrexate; purine
analogs such as fludarabine, 6-mercaptopurine, thiamiprine,
thioguanine; pyrimidine analogs such as ancitabine, azacitidine,
6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,
enocitabine, floxuridine, androgens such as calusterone,
dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;
demecolcine; diaziquone; elfomithine; elliptinium acetate;
etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine;
mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin;
phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide;
procarbazine; PSK.TM.; razoxane; sizofiran; spirogermanium;
tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethyla-mine;
urethan; vindesine; dacarbazine; mannomustine; mitobronitol;
mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C");
cyclophosphamide; thiotepa; taxanes, e.g. paclitaxel (TAXOL.TM.,
Bristol-Myers Squibb Oncology, Princeton, N.J.) and docetaxel
(TAXOTERE.TM., Rhone-Poulenc Rorer, Antony, France); retinoic acid;
esperamicins; capecitabine; and pharmaceutically acceptable salts,
acids or derivatives of any of the above. Also included as suitable
chemotherapeutic cell conditioners are anti-hormonal agents that
act to regulate or inhibit hormone action on tumors such as
anti-estrogens including for example tamoxifen (Nolvadex.TM.),
raloxifene, aromatase inhibiting 4(5)-imidazoles,
4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone,
and toremifene (Fareston); and anti-androgens such as flutamide,
nilutamide, bicalutamide, leuprolide, and goserelin; chlorambucil;
gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum
analogs such as cisplatin and carboplatin; vinblastine; platinum;
etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;
vincristine; vinorelbine; navelbine; novantrone; teniposide;
daunomycin; aminopterin; xeloda; ibandronate; camptothecin-11
(CPT-11); topoisomerase inhibitor RFS 2000; difluoromethylornithine
(DMFO). Where desired, the compounds or pharmaceutical composition
of the present invention can be used in combination with commonly
prescribed anti-cancer drugs such as Herceptin.RTM., Avastin.RTM.,
Erbitux.RTM., Rituxan.RTM., Taxol.RTM., Arimidex.RTM.,
Taxotere.RTM., and Velcade.RTM..
This invention further relates to a method for using the compounds
or pharmaceutical composition in combination with radiation therapy
in inhibiting abnormal cell growth or treating the
hyperproliferative disorder in the mammal. Techniques for
administering radiation therapy are known in the art, and these
techniques can be used in the combination therapy described herein.
The administration of the compound of the invention in this
combination therapy can be determined as described herein.
Radiation therapy can be administered through one of several
methods, or a combination of methods, including without limitation
external-beam therapy, internal radiation therapy, implant
radiation, stereotactic radiosurgery, systemic radiation therapy,
radiotherapy and permanent or temporary interstitial brachytherapy.
The term "brachytherapy," as used herein, refers to radiation
therapy delivered by a spatially confined radioactive material
inserted into the body at or near a tumor or other proliferative
tissue disease site. The term is intended without limitation to
include exposure to radioactive isotopes (e.g. At-211, I-131,
I-125, Y-90, Re-186, Re-188, Sm-153, Bi-212, P-32, and radioactive
isotopes of Lu). Suitable radiation sources for use as a cell
conditioner of the present invention include both solids and
liquids. By way of non-limiting example, the radiation source can
be a radionuclide, such as I-125, I-131, Yb-169, Ir-192 as a solid
source, I-125 as a solid source, or other radionuclides that emit
photons, beta particles, gamma radiation, or other therapeutic
rays. The radioactive material can also be a fluid made from any
solution of radionuclide(s), e.g., a solution of I-125 or I-131, or
a radioactive fluid can be produced using a slurry of a suitable
fluid containing small particles of solid radionuclides, such as
Au-198, Y-90. Moreover, the radionuclide(s) can be embodied in a
gel or radioactive micro spheres.
Without being limited by any theory, the compounds of the present
invention can render abnormal cells more sensitive to treatment
with radiation for purposes of killing and/or inhibiting the growth
of such cells. Accordingly, this invention further relates to a
method for sensitizing abnormal cells in a mammal to treatment with
radiation which comprises administering to the mammal an amount of
a compound of the present invention or pharmaceutically acceptable
salt, ester, prodrug, solvate, hydrate or derivative thereof, which
amount is effective is sensitizing abnormal cells to treatment with
radiation. The amount of the compound, salt, or solvate in this
method can be determined according to the means for ascertaining
effective amounts of such compounds described herein.
The compounds or pharmaceutical compositions of the present
invention can be used in combination with an amount of one or more
substances selected from anti-angiogenesis agents, signal
transduction inhibitors, and antiproliferative agents.
Anti-angiogenesis agents, such as MMP-2 (matrix-metalloprotienase
2) inhibitors, MMP-9 (matrix-metalloprotienase 9) inhibitors, and
COX-11 (cyclooxygenase 11) inhibitors, can be used in conjunction
with a compound of the present invention and pharmaceutical
compositions described herein. Examples of useful COX-II inhibitors
include CELEBREX.TM. (alecoxib), valdecoxib, and rofecoxib.
Examples of useful matrix metalloproteinase inhibitors are
described in WO 96/33172 (published Oct. 24, 1996), WO 96/27583
(published Mar. 7, 1996), European Patent Application No.
97304971.1 (filed Jul. 8, 1997), European Patent Application No.
99308617.2 (filed Oct. 29, 1999), WO 98/07697 (published Feb.
26,1998), WO 98/03516 (published Jan. 29, 1998), WO 98/34918
(published Aug. 13,1998), WO 98/34915 (published Aug. 13,1998), WO
98/33768 (published Aug. 6,1998), WO 98/30566 (published Jul. 16,
1998), European Patent Publication 606,046 (published Jul.
13,1994), European Patent Publication 931, 788 (published Jul. 28,
1999), WO 90/05719 (published May 31, 1990), WO 99/52910 (published
Oct. 21, 1999), WO 99/52889 (published Oct. 21, 1999), WO 99/29667
(published Jun. 17, 1999), PCT International Application No.
PCT/IB98/01113 (filed Jul. 21, 1998), European Patent Application
No. 99302232.1 (filed Mar. 25,1999), Great Britain Patent
Application No. 9912961.1 (filed Jun. 3, 1999), U.S. Provisional
Application No. 60/148,464 (filed Aug. 12, 1999), U.S. Pat. No.
5,863,949 (issued Jan. 26,1999), U.S. Pat. No. 5,861,510 (issued
Jan. 19, 1999), and European Patent Publication 780,386 (published
Jun. 25, 1997), all of which are incorporated herein in their
entireties by reference. Preferred MMP-2 and MMP-9 inhibitors are
those that have little or no activity inhibiting MMP-1. More
preferred, are those that selectively inhibit MMP-2 and/or AMP-9
relative to the other matrix-metalloproteinases (i. e., MAP-1,
MMP-3, MMP-4, MMP-5, MMP-6, MMP-7, MMP-8, MMP-10, MMP-11, MMP-12,
and MMP-13). Some specific examples of MMP inhibitors useful in the
present invention are AG-3340, RO 32-3555, and RS 13-0830.
The invention also relates to a method of and to a pharmaceutical
composition of treating a cardiovascular disease in a mammal which
comprises an amount of a compound of the present invention, or a
pharmaceutically acceptable salt, ester, prodrug, solvate, hydrate
or derivative thereof, or an isotopically-labeled derivative
thereof, and an amount of one or more therapeutic agents use for
the treatment of cardiovascular diseases.
Examples for use in cardiovascular disease applications are
anti-thrombotic agents, e.g., prostacyclin and salicylates,
thrombolytic agents, e.g., streptokinase, urokinase, tissue
plasminogen activator (TPA) and anisoylated
plasminogen-streptokinase activator complex (APSAC), anti-platelets
agents, e.g., acetyl-salicylic acid (ASA) and clopidrogel,
vasodilating agents, e.g., nitrates, calcium channel blocking
drugs, anti-proliferative agents, e.g., colchicine and alkylating
agents, intercalating agents, growth modulating factors such as
interleukins, transformation growth factor-beta and congeners of
platelet derived growth factor, monoclonal antibodies directed
against growth factors, anti-inflammatory agents, both steroidal
and non-steroidal, and other agents that can modulate vessel tone,
function, arteriosclerosis, and the healing response to vessel or
organ injury post intervention. Antibiotics can also be included in
combinations or coatings comprised by the invention. Moreover, a
coating can be used to effect therapeutic delivery focally within
the vessel wall. By incorporation of the active agent in a
swellable polymer, the active agent will be released upon swelling
of the polymer.
The compounds describe herein may be formulated or administered in
conjunction with liquid or solid tissue barriers also known as
lubricants. Examples of tissue barriers include, but are not
limited to, polysaccharides, polyglycans, seprafilm, interceed and
hyaluronic acid.
Medicaments which may be administered in conjunction with the
compounds described herein include any suitable drugs usefully
delivered by inhalation for example, analgesics, e.g. codeine,
dihydromorphine, ergotamine, fentanyl or morphine; anginal
preparations, e.g. diltiazem; antiallergics, e.g. cromoglycate,
ketotifen or nedocromil; anti-infectives, e.g. cephalosporins,
penicillins, streptomycin, sulphonamides, tetracyclines or
pentamidine; antihistamines, e.g. methapyrilene;
anti-inflammatories, e.g. beclomethasone, flunisolide, budesonide,
tipredane, triamcinolone acetonide or fluticasone; antitussives,
e.g. noscapine; bronchodilators, e.g. ephedrine, adrenaline,
fenoterol, formoterol, isoprenaline, metaproterenol, phenylephrine,
phenylpropanolamine, pirbuterol, reproterol, rimiterol, salbutamol,
salmeterol, terbutalin, isoetharine, tulobuterol, orciprenaline or
(+4-amino-3,5-dichloro-.alpha.-[[[6-[2-(2-pyridinyl)ethoxy]hexyl]-amino]m-
ethyl]benzenemethanol; diuretics, e.g. amiloride; anticholinergics
e.g. ipratropium, atropine or oxitropium; hormones, e.g. cortisone,
hydrocortisone or prednisolone; xanthines e.g. aminophylline,
choline theophyllinate, lysine theophyllinate or theophylline; and
therapeutic proteins and peptides, e.g. insulin or glucagon. It
will be clear to a person skilled in the art that, where
appropriate, the medicaments may be used in the form of salts (e.g.
as alkali metal or amine salts or as acid addition salts) or as
esters (e.g. lower alkyl esters) or as solvates (e.g. hydrates) to
optimize the activity and/or stability of the medicament.
Other exemplary therapeutic agents useful for a combination therapy
include but are not limited to agents as described above, radiation
therapy, hormone antagonists, hormones and their releasing factors,
thyroid and antithyroid drugs, estrogens and progestins, androgens,
adrenocorticotropic hormone; adrenocortical steroids and their
synthetic analogs; inhibitors of the synthesis and actions of
adrenocortical hormones, insulin, oral hypoglycemic agents, and the
pharmacology of the endocrine pancreas, agents affecting
calcification and bone turnover: calcium, phosphate, parathyroid
hormone, vitamin D, calcitonin, vitamins such as water-soluble
vitamins, vitamin B complex, ascorbic acid, fat-soluble vitamins,
vitamins A, K, and E, growth factors, cytokines, chemokines,
muscarinic receptor agonists and antagonists; anticholinesterase
agents; agents acting at the neuromuscular junction and/or
autonomic ganglia; catecholamines, sympathomimetic drugs, and
adrenergic receptor agonists or antagonists; and
5-hydroxytryptamine (5-HT, serotonin) receptor agonists and
antagonists.
Therapeutic agents can also include agents for pain and
inflammation such as histamine and histamine antagonists,
bradykinin and bradykinin antagonists, 5-hydroxytryptamine
(serotonin), lipid substances that are generated by
biotransformation of the products of the selective hydrolysis of
membrane phospholipids, eicosanoids, prostaglandins, thromboxanes,
leukotrienes, aspirin, nonsteroidal anti-inflammatory agents,
analgesic-antipyretic agents, agents that inhibit the synthesis of
prostaglandins and thromboxanes, selective inhibitors of the
inducible cyclooxygenase, selective inhibitors of the inducible
cyclooxygenase-2, autacoids, paracrine hormones, somatostatin,
gastrin, cytokines that mediate interactions involved in humoral
and cellular immune responses, lipid-derived autacoids,
eicosanoids, .beta.-adrenergic agonists, ipratropium,
glucocorticoids, methylxanthines, sodium channel blockers, opioid
receptor agonists, calcium channel blockers, membrane stabilizers
and leukotriene inhibitors.
Additional therapeutic agents contemplated herein include
diuretics, vasopressin, agents affecting the renal conservation of
water, rennin, angiotensin, agents useful in the treatment of
myocardial ischemia, anti-hypertensive agents, angiotensin
converting enzyme inhibitors, .beta.-adrenergic receptor
antagonists, agents for the treatment of hypercholesterolemia, and
agents for the treatment of dyslipidemia.
Other therapeutic agents contemplated include drugs used for
control of gastric acidity, agents for the treatment of peptic
ulcers, agents for the treatment of gastroesophageal reflux
disease, prokinetic agents, antiemetics, agents used in irritable
bowel syndrome, agents used for diarrhea, agents used for
constipation, agents used for inflammatory bowel disease, agents
used for biliary disease, agents used for pancreatic disease.
Therapeutic agents used to treat protozoan infections, drugs used
to treat Malaria, Amebiasis, Giardiasis, Trichomoniasis,
Trypanosomiasis, and/or Leishmaniasis, and/or drugs used in the
chemotherapy of helminthiasis. Other therapeutic agents include
antimicrobial agents, sulfonamides, trimethoprim-sulfamethoxazole
quinolones, and agents for urinary tract infections, penicillins,
cephalosporins, and other, .beta.-Lactam antibiotics, an agent
comprising an aminoglycoside, protein synthesis inhibitors, drugs
used in the chemotherapy of tuberculosis, mycobacterium avium
complex disease, and leprosy, antifungal agents, antiviral agents
including nonretroviral agents and antiretroviral agents.
Examples of therapeutic antibodies that can be combined with a
subject compound include but are not limited to anti-receptor
tyrosine kinase antibodies (cetuximab, panitumumab, trastuzumab),
anti CD20 antibodies (rituximab, tositumomab), and other antibodies
such as alemtuzumab, bevacizumab, and gemtuzumab.
Moreover, therapeutic agents used for immunomodulation, such as
immunomodulators, immunosuppressive agents, tolerogens, and
immunostimulants are contemplated by the methods herein. In
addition, therapeutic agents acting on the blood and the
blood-forming organs, hematopoietic agents, growth factors,
minerals, and vitamins, anticoagulant, thrombolytic, and
antiplatelet drugs.
Further therapeutic agents that can be combined with a subject
compound may be found in Goodman and Gilman's "The Pharmacological
Basis of Therapeutics" Tenth Edition edited by Hardman, Limbird and
Gilman or the Physician's Desk Reference, both of which are
incorporated herein by reference in their entirety.
The compounds described herein can be used in combination with the
agents disclosed herein or other suitable agents, depending on the
condition being treated. Hence, in some embodiments the compounds
of the invention will be co-administer with other agents as
described above. When used in combination therapy, the compounds
described herein may be administered with the second agent
simultaneously or separately. This administration in combination
can include simultaneous administration of the two agents in the
same dosage form, simultaneous administration in separate dosage
forms, and separate administration. That is, a compound described
herein and any of the agents described above can be formulated
together in the same dosage form and administered simultaneously.
Alternatively, a compound of the present invention and any of the
agents described above can be simultaneously administered, wherein
both the agents are present in separate formulations. In another
alternative, a compound of the present invention can be
administered just followed by and any of the agents described
above, or vice versa. In the separate administration protocol, a
compound of the present invention and any of the agents described
above may be administered a few minutes apart, or a few hours
apart, or a few days apart.
The examples and preparations provided below further illustrate and
exemplify the compounds of the present invention and methods of
preparing such compounds. It is to be understood that the scope of
the present invention is not limited in any way by the scope of the
following examples and preparations. In the following examples
molecules with a single chiral center, unless otherwise noted,
exist as a racemic mixture. Those molecules with two or more chiral
centers, unless otherwise noted, exist as a racemic mixture of
diastereomers. Single enantiomers/diastereomers may be obtained by
methods known to those skilled in the art.
EXAMPLES
Example 1: Synthesis of
3-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)--
8-methyl-2-o-tolylisoquinolin-1(2H)-one (Compound 1613) (method
A).
##STR00312## ##STR00313##
A solution of 2-amino-6-methylbenzoic acid (1601) (106.5 g, 705
mmol) in H.sub.2O (200 mL) was cooled to 0-5.degree. C., con. HCl
(250 mL) was added slowly. The solution was stirred for 15 min at
0-5.degree. C. A solution of sodium nitrite (58.4 g, 6.85 mol) in
H.sub.2O (120 mL) was added dropwise at 0-5.degree. C., and the
resulting mixture was stirred for 30 min. Then above solution was
added to a solution of KI (351 g, 2.11 mol) in H.sub.2O (200 mL),
and the resulting mixture was stirred at RT for 16 h. The solution
was poured into ice water (2000 mL) and extracted with ethyl
acetate (3.times.1000 mL). The combined organic layer was washed
with aqueous NaOH (15%, 3.times.200 mL). The aqueous layer was
acidified to PH=1, and extracted with ethyl acetate (3.times.1000
mL). The combined organic layer was dried over Na.sub.2SO.sub.4 and
filtered. The filtrate was concentrated in vacuo to afford the
desired product, 2-iodo-6-methylbenzoic acid (1602) (145 g, 79%
yield) as a yellow solid
To a stirred mixture of 2-iodo-6-methylbenzoic acid (1602) (105 g,
400 mmol), Pd(OAc).sub.2 (27 g, 120 mmol) and PPh.sub.3 (63 g 240
mol) in THF (1000 mL) at RT, tributyl(vinyl)tin (152 g, 480 mmol)
was added. The resulting mixture was heated to reflux overnight.
The mixture was allowed to cool to RT, filtered through silica gel
(10 g), and then concentrated in vacuo. The residue was poured into
ice water (1000 mL) and extracted with ethyl acetate (3.times.1000
mL). The combined organic layer was washed with aqueous NaOH (15%,
5.times.200 mL). The combined aqueous layer was acidified to PH=1,
extracted with ethyl acetate (3.times.1000 mL). The combined
organic layer was dried over Na.sub.2SO.sub.4 and filtered. The
filtrate was concentrated in vacuo to afford the desired product,
2-methyl-6-vinylbenzoic acid (1603) (61 g, 95% yield) as a yellow
solid.
A mixture of 2-methyl-6-vinylbenzoic acid (1603) (56 g, 350 mmol)
and thionyl chloride (208 g, 1750 mmol) in toluene (400 mL) was
stirred at reflux for 2 h. The mixture was concentrated in vacuo to
afford the desired product, 2-methyl-6-vinylbenzoyl chloride (1604)
(63 g, 95% yield) as a yellow oil. The product obtained was used
directly in the next step without purification.
A mixture of o-toluidine (45 g, 420 mmol) and Triethylamine (71 g,
70 mmol) in CH.sub.2Cl.sub.2 (300 mL) was stirred for 10 min at RT.
To this mixture, 2-methyl-6-vinylbenzoyl chloride (1604) (63 g, 35
mmol) was added, and the resulting mixture was stirred at RT for 30
min. The solution was poured into water (300 mL) and extracted with
CH.sub.2Cl.sub.2 (3.times.200 mL), dried over Na.sub.2SO.sub.4 and
filtered. The filtrate was concentrated in vacuo to afford the
crude product. The crude product was suspended in IPE (isopropyl
ether) (300 mL), stirred at reflux for 30 min, and then cooled to
0-5.degree. C. The precipitate was collected by filtration and
further dried in vacuo to afford the desired product,
2-methyl-N-o-tolyl-6-vinylbenzamide (1605) (81 g, 80% yield) as a
yellow solid.
To a solution of 2-methyl-N-o-tolyl-6-vinylbenzamide (1605) (80 g,
320 mmol) in DMF (250 mL) at RT, NaH (60% in mineral oil, 25.6 g,
640 mmol) was slowly added and the resulting mixture was stirred at
RT for 30 min. To this mixture, ethyl chloroacetate (78 g, 640
mmol) was added and the resulting mixture was stirred at RT for 2
h. The solution was poured into water (500 mL) and extracted with
ethyl acetate (3.times.200 mL), dried over Na.sub.2SO.sub.4 and
filtered. The filtrate was concentrated in vacuo. The crude product
was suspended in MeOH (160 mL), stirred at reflux for 10 min, and
then cooled to 0-5.degree. C. The precipitate was collected by
filtration and further dried in vacuo to afford the desired
product, ethyl 2-(2-methyl-N-o-tolyl-6-vinylbenzamido)acetate
(1606) (67 g, 62% yield) as a white solid.
To a stirred mixture of ethyl
2-(2-methyl-N-o-tolyl-6-vinylbenzamido)acetate (1606) (67 g, 200
mmol) in 1,4-dioxane (300 mL) and H.sub.2O (100 mL) at RT, Osmium
tetroxide (20 mg) was added was and stirred at RT for 30 min. To
this mixture, sodium periodate (86 g, 400 mmol) was added and the
resulting mixture was stirred at RT for 16 h. The reaction mixture
was filtered through silica gel (10 g), the filtrate was extracted
with ethyl acetate (3.times.200 mL). The combined organic layers
were washed with brine (100 mL), dried over Na.sub.2SO.sub.4 and
filtered. The filtrate was concentrated in vacuo and the residue
was further dried in vacuo to afford the desired product, ethyl
2-(2-formyl-6-methyl-N-o-tolylbenzamido)acetate (1607) (38 g, 57%
yield) as a yellow solid.
To a stirred solution of ethyl
2-(2-formyl-6-methyl-N-o-tolylbenzamido)acetate (1607) (38 g, 112
mmol) in EtOH (200 mL) and ethyl acetate (100 mL) at RT, cesium
carbonate (22 g, 112 mmol) was added. The resulting mixture was
degassed and back-filled with argon three times and then stirred at
50.degree. C. for 5 h. The mixture was allowed to cool to RT,
filtered through silica gel (10 g), and the filtrate was
concentrated in vacuo. The residue was poured into H.sub.2O (200
mL), extracted with ethyl acetate (3.times.200 mL). The combined
organic layer was washed with brine (50 mL), dried over
Na.sub.2SO.sub.4 and filtered. The filtrate was concentrated in
vacuo. The crude product was suspended in IPE (120 mL), heated to
reflux for 10 min, and then cooled to 0-5.degree. C. The
precipitate was collected by filtration and further dried in vacuo
to afford the desired product, ethyl
8-methyl-1-oxo-2-o-tolyl-1,2-dihydroisoquinoline-3-carboxylate
(1608) (28 g, 77% yield) as a white solid.
To a stirred solution of lithium aluminum hydride (8.28 g, 218 mol)
in anhydrous THF (500 mL) at -78.degree. C. under a nitrogen
atmosphere, ethyl
8-methyl-1-oxo-2-o-tolyl-1,2-dihydroisoquinoline-3-carboxylate
(1608) (28 g, 87 mmol) was slowly added over a 10 min period of
time. The resulting mixture was allowed to warm to -30.degree. C.,
stirred for 30 min and TLC showed the completion of the reaction.
Then the mixture was cooled to -78.degree. C., and water (50 mL)
was slowly added. The mixture was allowed to warm to RT, filtered
through silica gel (10 g), and the filtrate was concentrated in
vacuo. The crude product was poured into H.sub.2O (200 mL) and
extracted with ethyl acetate (3.times.200 mL). The combined organic
layer was washed with brine (50 mL), dried over Na.sub.2SO.sub.4
and filtered. The filtrate was concentrated in vacuo. The crude
product was suspended in ethyl acetate (30 mL) and stirred for 10
min. The solid was collected by filtration and further dried in
vacuo to afford the desired product,
3-(hydroxymethyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one (1609)
(22 g, 92% yield) as a white solid.
PBr.sub.3 (25.6 g, 95 mmol) was slowly added to a stirred solution
of DMF (11.5 g, 158 mol) in acetonitrile (200 mL) at 0.degree. C.,
and the resulting mixture was stirred at OC for 30 min.
3-(Hydroxymethyl)-8-methyl-2-o-tolylisoquinolin-1-(2H)-one (1609)
(22 g, 78.8 mmol) was slowly added. Then the reaction mixture was
allowed to warm to RT and stirred for 30 min. Saturated aqueous
NaHCO.sub.3 solution (50 mL) was slowly added and extracted with
ethyl acetate (3.times.200 mL). The combined organic layer was
washed with brine, dried over Na.sub.2SO.sub.4 and filtered. The
filtrate was concentrated in vacuo. The crude product was suspended
in IPE (50 mL) and then stirred for 10 min. The precipitate was
collected by filtration and further dried in vacuo to afford the
desired product,
3-(bromomethyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one (1610) (21
g, 80% yield) as a white solid.
3-Iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (108) (10.8 g, 41.4
mmol) and potassium tert-butoxide (4.4 g, 40 mmol) were dissolved
in anhydrous DMF (150 mL) and stirred at RT for 30 min.
3-(Bromomethyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one (1610)
(13.7 g, 40 mmol) was added. The resulting mixture was stirred at
RT for 30 min, poured into ice water (300 mL) and then extracted
with ethyl acetate (3.times.200 mL). The combined organic layer was
washed with brine (50 mL), dried over Na.sub.2SO.sub.4 and
filtered. The filtrate was concentrated to about 100 ml in vacuo,
the precipitate was collected by filtration to afford the first
batch of desired product,
3-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-8-methyl-2-o--
tolylisoquinolin-1(2H)-one (1611) (12 g, 60% yield) as a white
solid. The filtrate was concentrated in vacuo and the residue was
purified by flash column chromatography on silica gel (2-20%
MeOH/DCM) to afford the second batch of desired product,
3-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-8-methyl-2-o--
tolylisoquinolin-1(2H)-one (1611) (6 g, 30% yield) as a white
solid.
3-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-8-methyl-2-o--
tolylisoquinolin-1(2H)-one (1611) (13 g, 24.9 mmol) and
3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (1612)(6.6 g,
30 mmol) were dissolved in DMF-EtOH-H.sub.2O (120 mL, 40 mL, 40
mL). Pd(OAc).sub.2 (1.684 g, 7.5 mmol), PPh.sub.3 (3.935 g 15 mmol)
and Na.sub.2CO.sub.3 (13.25 g 125 mmol) were added sequentially.
The resulting mixture was degassed and back-filled with argon three
times and then stirred at 100.degree. C. for 1 h. The mixture was
allowed to cool to RT, filtered through silica gel (10 g) and
concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel (2-20% MeOH/DCM) to afford the product
(1613) (9 g, 76% yield) as a slight yellow solid. Then above
product was suspended in EtOH (100 mL) and heated to reflux for 30
min. The mixture was allowed to cool to RT, and the solid was
collected by filtration. The solid was then suspended in EA (100
mL) and stirred overnight. The precipitate was collected by
filtration and further dried in vacuo to afford the desired
product,
3-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)--
8-methyl-2-o-tolylisoquinolin-1(2H)-one (1613)(8.4 g, 69% yield) as
a white solid.
Example 2: Synthesis of
3-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)--
8-methyl-2-o-tolylisoquinolin-1(2H)-one (Compound 1613) (method
B).
##STR00314##
3-(3-Methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (1701)(964
mg, 4 mmol) and potassium tert-butoxide (0.44 g, 4 mmol) were
dissolved in anhydrous DMF (150 mL) and stirred at RT for 30 min.
3-(Bromomethyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one (1610)
(1.37 g, 4.0 mmol) was added. The resulting mixture was stirred at
RT for 30 min, poured into ice water (30 mL) and then extracted
with ethyl acetate (3.times.50 mL). The combined organic layer was
washed with brine (25 mL), dried over Na.sub.2SO.sub.4 and
filtered. The filtrate was concentrated in vacuo and the residue
was purified by flash column chromatography on silica gel (2-20%
MeOH/DCM) to afford the desired product,
3-((4-amino-3-(3-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl-
)methyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one (1702) (1.4 g, 70%
yield) as a white solid.
To a solution of
3-((4-amino-3-(3-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)--
8-methyl-2-o-tolylisoquinolin-1(2H)-one (1702)(100 mg, 0.2 mmol) in
CH.sub.2Cl.sub.2 (20 mL) at -78.degree. C. under a nitrogen
atmosphere, BBr.sub.3 (1 mL) was added and the resulting mixture
was stirred at -78.degree. C. for 3 h. The mixture was allowed to
warm to RT, poured into ice-water (200 mL) and extracted with ethyl
acetate (3.times.50 mL). The combined organic layer was washed with
brine (20 mL), dried over Na.sub.2SO.sub.4 and filtered. The
filtrate was concentrated in vacuo and the residue was purified by
flash column chromatography on silica gel (10-50%
MeOH/CH.sub.2Cl.sub.2) to afford the desired product,
3-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)--
8-methyl-2-o-tolylisoquinolin-1(2H)-one (1613)(87 mg, 91% yield) as
a white solid.
Example 3: Synthesis of
(R)-3-((4-amino-3-(3-hydroxybut-1-ynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-
methyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one (Compound
1802).
##STR00315##
3-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-8-methyl-2-o--
tolylisoquinolin-1(2H)-one (1611) (522 mg, 1 mmol) and
(R)-but-3-yn-2-ol (84 mg, 1.2 mmol) were dissolved in anhydrous THF
(40 mL). The mixture was degassed and back-filled with nitrogen
three times. Pd(PPh.sub.3).sub.2Cl.sub.2 (12 mg, 0.1 mmol), CuI (47
mg 0.25 mmol) and (i-Pr).sub.2NH (505 mg, 5 mmol) were added
sequentially. The resulting mixture was degassed and back-filled
with argon three times and then stirred at reflux for 4 h. The
mixture was allowed to cool to RT, filtered through silica gel (10
g) and concentrated in vacuo. The residue was purified by flash
column chromatography on silica gel (2-20% MeOH/DCM) to afford the
product, 3
(R)-3-((4-amino-3-(3-hydroxybut-1-ynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-
methyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one (1802) (324 mg, 70%
yield) as a slightly yellow solid.
Example 4: Synthesis of
3-((6-amino-9H-purin-9-yl)methyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one
(Compound 1902).
##STR00316##
9H-Purin-6-amine (1901)(540 mg, 4.0 mmol) was dissolved in
anhydrous DMF (20 mL). NaH (60% in mineral oil, 160 mg, 4.0 mmol)
was added and the resulting mixture was stirred at RT for 30 min.
3-(Bromomethyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one (1610)(1.37
g, 4.0 mmol) was added. The reaction mixture was stirred at RT for
30 min, poured into ice-water (30 mL) and then extracted with ethyl
acetate (3.times.50 mL). The combined organic layer was washed with
brine (25 mL), dried over Na.sub.2SO.sub.4 and filtered. The
filtrate was concentrated in vacuo and the residue was purified by
flash column chromatography on silica gel (2-20% MeOH/DCM) to
afford the desired product,
34(6-amino-9H-purin-9-yl)methyl)-8-methyl-2-o-tolylisoquinolin-1-
(2H)-one (1902)(1.1 g, 70% yield) as a white solid.
Example 5: Synthesis of
3-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)--
2-isopropyl-8-methylisoquinolin-1(2H)-one (Compound 2013).
##STR00317## ##STR00318##
To a stirred mixture of 2-iodo-6-methylbenzoic acid (1602) (105 g,
400 mmol), Pd(OAc).sub.2 (27 g, 120 mmol) and PPh.sub.3 (63 g 240
mol) in THF (1000 mL) at RT, tributyl(vinyl)tin (152 g, 480 mmol)
was added. The resulting mixture was heated to reflux overnight.
The mixture was allowed to cool to RT, filtered through silica gel
(10 g), and then concentrated in vacuo. The residue was poured into
ice water (1000 mL) and extracted with ethyl acetate (3.times.1000
mL). The combined organic layer was washed with aqueous NaOH (15%,
5.times.200 mL). The combined aqueous layer was acidified to PH=1,
extracted with ethyl acetate (3.times.1000 mL). The combined
organic layer was dried over Na.sub.2SO.sub.4 and filtered. The
filtrate was concentrated in vacuo to afford the desired product,
2-methyl-6-vinylbenzoic acid (1603) (61 g, 95% yield) as a yellow
solid.
A mixture of 2-methyl-6-vinylbenzoic acid (1603) (56 g, 350 mmol)
and thionyl chloride (208 g, 1750 mmol) in toluene (400 mL) was
stirred at reflux for 2 h. The mixture was concentrated in vacuo to
afford the desired product, 2-methyl-6-vinylbenzoyl chloride (1604)
(63 g, 95% yield) as a yellow oil. The product obtained was used
directly in the next step without purification.
Propan-2-amine (2001)(59 g, 1.0 mol) and ethyl chloroacetate (122
g, 1.0 mol) were dissolved in toluene (200 mL) and the mixture was
stirred at reflux for 2 h. The reaction mixture was allowed to cool
to RT, poured into ice-water (500 mL) and extracted with ethyl
acetate (3.times.250 mL). The combined organic layer was washed
with brine (50 mL), dried over Na.sub.2SO.sub.4 and filtered. The
filtrate was concentrated in vacuo and the residue was purified by
flash column chromatography on silica gel (10-50% EA/PE) to afford
the product, ethyl 2-(isopropylamino)acetate (2002) (70 g, 51%
yield) as an oil.
Ethyl 2-(isopropylamino)acetate (2002) (14.5 g, 100 mmol) and
triethylamine (200 g, 200 mmol) were dissolved in CH.sub.2Cl.sub.2
(300 mL) and the mixture was stirred for 10 min at RT.
2-Methyl-6-vinylbenzoyl chloride (1604) (18 g, 100 mmol) was added,
and the resulting mixture was stirred at RT for 30 min. The
reaction mixture was poured into water (300 mL) and extracted with
CH.sub.2Cl.sub.2 (3.times.200 mL). The combined organic layer was
washed with brine (50 mL), dried over Na.sub.2SO.sub.4 and
filtered. The filtrate was concentrated in vacuo to afford the
crude product. The crude product was suspended in IPE (isopropyl
ether) (300 mL), stirred at reflux for 30 min, and then cooled to
0-5.degree. C. The precipitate was collected by filtration and
further dried in vacuo to afford the desired product, ethyl
2-(N-isopropyl-2-methyl-6-vinylbenzamido)acetate (2003) (14.5 g,
50% yield) as a yellow solid.
To a stirred solution of ethyl
2-(N-isopropyl-2-methyl-6-vinylbenzamido)acetate (2003) (14.0 g,
48.0 mmol) in 1,4-dioxane (100 mL) and H.sub.2O (30 mL), Osmium
tetroxide (20 mg) was added and the resulting mixture was stirred
at RT for 30 min. To this mixture, sodium periodate (22 g, 100
mmol) was added and then stirred at RT for 16 h. The reaction
mixture was filtered through silica gel (10 g), the filtrate was
extracted with ethyl acetate (3.times.200 mL). The combined organic
layer was washed with brine (50 mL), dried over Na.sub.2SO.sub.4
and filtered. The filtrate was concentrated in vacuo and the
residue was further dried in vacuo to afford the desired product,
ethyl 2-(2-formyl-N-isopropyl-6-methylbenzamido)acetate (2004)
(8.33 g, 57% yield) as a yellow solid.
To a stirred solution of ethyl
2-(2-formyl-N-isopropyl-6-methylbenzamido)acetate (2004) (8.3 g,
28.0 mmol) in EtOH (100 mL) and ethyl acetate (50 mL) at RT, cesium
carbonate (5.9 g, 30 mmol) was added. The resulting mixture was
degassed and back-filled with argon three times and then stirred at
50.degree. C. for 5 h. The mixture was allowed to cool to RT,
filtered through silica gel (10 g), and the filtrate was
concentrated in vacuo. The residue was poured into H.sub.2O (200
mL), extracted with ethyl acetate (3.times.200 mL). The combined
organic layer was washed with brine (50 mL), dried over
Na.sub.2SO.sub.4 and filtered. The filtrate was concentrated in
vacuo. The crude product was suspended in IPE (120 mL), stirred at
reflux for 10 min, and then cooled to 0-5.degree. C. The
precipitate was collected by filtration and further dried in vacuo
to afford the desired product, ethyl
2-isopropyl-8-methyl-1-oxo-1,2-dihydroisoquinoline-3-carboxylate
(2005) (5.35 g, 70% yield) as a white solid.
To a stirred solution of lithium aluminum hydride (2.88 g, 76 mol)
in anhydrous THF (200 mL) at -78.degree. C. under a nitrogen
atmosphere, ethyl
2-isopropyl-8-methyl-1-oxo-1,2-dihydroisoquinoline-3-carboxylate
(2005) (5.2 g, 19 mmol) was slowly added over a 10 min period of
time. The resulting mixture was allowed to warm to -30.degree. C.,
stirred for 30 min and TLC showed the completion of the reaction.
Then the mixture was cooled to -78.degree. C., and water (50 mL)
was slowly added. The mixture was allowed to warm to RT, filtered
through silica gel (10 g), and the filtrate was concentrated in
vacuo. The crude product was poured into H.sub.2O (200 mL) and
extracted with ethyl acetate (3.times.200 mL). The combined organic
layer was washed with brine (50 mL), dried over Na.sub.2SO.sub.4
and filtered. The filtrate was concentrated in vacuo. The crude
product was suspended in ethyl acetate (30 mL) and stirred for 10
min. The solid was collected by filtration and further dried in
vacuo to afford the desired product,
3-(hydroxymethyl)-2-isopropyl-8-methylisoquinolin-1(2H)-one (2006)
(3.51 g, 80% yield) as a white solid.
To a solution of
3-(hydroxymethyl)-2-isopropyl-8-methylisoquinolin-1(2H)-one (2006)
(1.61 g, 7.0 mmol) in CH.sub.2Cl.sub.2, PPh.sub.3 (3.67 g, 14.0
mmol) was added and the mixture was stirred at RT for 30 min. The
mixture was cooled to 0.degree. C., and CBr.sub.4 (4.64 g, 14.0
mmol) was added in portions. The resulting mixture was stirred from
0.degree. C. to RT for 30 min, and then concentrated in vacuo. The
crude product was purified by flash column chromatography on silica
gel (30-50% EA/PE) to afford the desired product,
3-(bromomethyl)-2-isopropyl-8-methylisoquinolin-1(2H)-one (2007)
(1.65 g, 80% yield) as a white solid.
A mixture of 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (108) (1.3
g, 5 mmol) and potassium tert-butoxide (0.55 g, 5 mmol) in
anhydrous DMF (20 mL) was stirred at RT for 30 min and then
3-(bromomethyl)-2-isopropyl-8-methylisoquinolin-1(2H)-one (2007)
(1.47 g, 5 mmol) was added. The resulting mixture was stirred at RT
for 30 min, poured into ice-water (30 mL) and then extracted with
ethyl acetate (3.times.50 mL). The combined organic layer was
washed with brine (25 mL), dried over Na.sub.2SO.sub.4 and
filtered. The filtrate was concentrated in vacuo, and the residue
was purified by flash column chromatography on silica gel (2-20%
MeOH/DCM) to afford the desired product,
3-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-is-
opropyl-8-methylisoquinolin-1(2H)-one (2008) (1.66 g, 70% yield) as
a white solid.
To a stirred mixture of
3-((4-Amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-isopropyl-8-
-methylisoquinolin-1(2H)-one (2008) (95 mg, 0.2 mmol) and
3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (66 mg, 0.3
mmol) in DMF-EtOH-H.sub.2O (3:1:1, 20 mL), Pd(OAc).sub.2 (16 mg,
0.075 mmol), PPh.sub.3 (39.3 mg 0.15 mmol) and Na.sub.2CO.sub.3
(132 mg, 1.25 mmol) were added sequentially. The resulting mixture
was degassed and back-filled with argon three times and then
stirred at 100.degree. C. for 1 h. The mixture was allowed to cool
to RT, filtered through silica gel (10 g) and concentrated in
vacuo. The residue was purified by flash column chromatography on
silica gel (2-20% MeOH/DCM) to afford the product,
34(4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-
methyl)-2-isopropyl-8-methylisoquinolin-1(2H)-one (2009) (53 mg,
61% yield) as a slightly yellow solid.
Example 6: Synthesis of
8-methyl-3-((methyl(9H-purin-6-yl)amino)methyl)-2-o-tolylisoquinolin-1(2H-
)-one.
##STR00319##
3-(Bromomethyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one (342 mg,
1.0 mmol) 1610 was dissolved in methylamine solution (100 mL) and
stirred for 2 h. The mixture was poured into ice-water (200 mL) and
extracted with ethyl acetate (3.times.50 mL). The combined organic
layer was washed with brine (20 mL), dried over Na.sub.2SO.sub.4
and filtered. The filtrate was concentrated in vacuo to afford the
desired product,
8-methyl-3-((methylamino)methyl)-2-o-tolylisoquinolin-1(2H)-one
(4001) (250 mg, 86% yield) as a yellow solid. The product obtained
was used directly in the next step without purification.
8-Methyl-3-((methylamino)methyl)-2-o-tolylisoquinolin-1(2H)-one
(233 mg, 0.8 mmol) (4001) and
6-chloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purine (4002) (238 mg, 1.0
mmol) were dissolved in EtOH (50 mL) and the resulting mixture was
stirred at reflux for 2 h. The mixture was allowed to cool to RT,
and concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel (2-20% MeOH/DCM) to afford the
product,
8-Methyl-3-((methyl(9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-yl)a-
mino)methyl)-2-o-tolylisoquinolin-1(2H)-one (4003) (200 mg, 51%
yield) as a slight yellow solid.
8-Methyl-3-((methyl(9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-yl)amino)meth-
yl)-2-o-tolylisoquinolin-1(2H)-one (4003) (180 mg 0.36 mmol) was
dissolved in MeOH (HCl) (50 mL) and the mixture was stirred at RT
for 2 h. Aqueous NaHCO.sub.3 solution was added to the reaction
mixture and the pH value was adjusted to 9. The mixture was
filtered and the filtrate was concentrated in vacuo to afford the
desired product,
8-methyl-3-((methyl(9H-purin-6-yl)amino)methyl)-2-o-tolylisoquinolin-1(2H-
)-one (4004) (80 mg, 54% yield) as a yellow solid.
Example 7: Synthesis of
3-(1-(9H-purin-6-ylamino)ethyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one.
##STR00320## ##STR00321##
To a stirred solution of
3-(hydroxymethyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one 1609
(2.79 g, 10 mmol) in CH.sub.2Cl.sub.2 (200 mL), MnO.sub.2 (5 g) was
added and the resulting mixture was stirred at reflux for 3 h. The
mixture was allowed to cool to RT, and concentrated in vacuo. The
residue was purified by flash column chromatography on silica gel
(10-50% EA/PE) to afford the product,
8-methyl-1-oxo-2-o-tolyl-1,2-dihydroisoquinoline-3-carbaldehyde
4101 (2.5 g, 90% yield) as a white solid.
8-Methyl-1-oxo-2-o-tolyl-1,2-dihydroisoquinoline-3-carbaldehyde
4101 (2.4 g, 8.6 mmol) was dissolved in anhydrous THF (280 mL) and
cooled to -78.degree. C. under a nitrogen atmosphere. Methyl MgBr
(2 M, 5 mL, 10 mmol) was added slowly, and the resulting mixture
was stirred at -78.degree. C. for 2 h. H.sub.2O (5 mL) was added
and then the solution was poured into ice-water (200 mL) and
extracted with ethyl acetate (3.times.50 mL). The combined organic
layer was washed with brine, dried over Na.sub.2SO.sub.4 and
filtered. The filtrate was concentrated in vacuo, and the residue
product was purified by flash column chromatography on silica gel
(10-50% EA/PE) to afford the product,
3-(1-hydroxyethyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one 4102
(1.8 g, 71% yield) as a white solid.
To a solution of
3-(1-hydroxyethyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one 4102
(1.6 g, 5.5 mmol) in CH.sub.2Cl.sub.2, PPh.sub.3 (2.88 g, 11.0
mmol) was added and the resulting mixture was stirred at RT for 30
min. Then CBr.sub.4 (3.64 g, 11.0 mmol) was added in portions to
the mixture at 0.degree. C. The resulting mixture was allowed to
warm to RT, stirred for 30 min, and concentrated in vacuo. The
crude product was purified by flash column chromatography on silica
gel (30-50% EA/PE) to afford the desired product,
3-(1-bromoethyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one 4103 (1.8
g, 91% yield) as a white solid.
To a stirred solution of
9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine 4103 (436 mg 2 mmol)
in anhydrous DMF (10 mL), NaH (60% in mineral oil, 77 mg, 2 mmol)
was added and the mixture was stirred for 30 min.
3-(1-Bromoethyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one 4104 (700
mg, 2 mmol) was added. The mixture was stirred for 2 h, poured into
ice-water (200 mL) and extracted with ethyl acetate (3.times.50
mL). The combined organic layer was washed with brine (20 mL),
dried over Na.sub.2SO.sub.4 and filtered. The filtrate was
concentrated in vacuo and the residue was purified by flash column
chromatography on silica gel (10-50% MeOH/DCM) to afford the
product,
8-methyl-3-(1-(9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-ylamino)ethyl)-2-o-
-tolylisoquinolin-1(2H)-one 4105 (500 mg, 51% yield) as a white
solid.
8-Methyl-3-(1-(9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-ylamino)ethyl)-2-o-
-tolylisoquinolin-1(2H)-one 4105 (180 mg, 0.36 mmol) was dissolved
in MeOH (HCl) (50 mL) and stirred for 2 h. Aqueous NaHCO.sub.3
solution was added to the reaction mixture and the pH value was
adjusted to 9. The mixture was then filtered and the filtrate was
concentrated in vacuo to afford the desired product,
3-(1-(9H-purin-6-ylamino)ethyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one
4106 (80 mg, 54% yield) as a yellow solid.
Example 8: Synthesis of
3-(4-amino-1-((8-methyl-1-oxo-2-o-tolyl-1,2-dihydroisoquinolin-3-yl)methy-
l)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-5-fluorophenyl dihydrogen
phosphate.
##STR00322##
3-((4-Amino-3-(3-fluoro-5-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl-
)methyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one 4301 (250 mg, 0.5
mmol) was dissolved in anhydrous THF (15 mL) in a round bottom
flask in dark (covered by aluminum foil) and cooled to 0.degree. C.
under an argon atmosphere. CBr.sub.4 (498 mg, 1.5 mmol) was added
followed by diethylphosphite (129 .mu.L, 1.0 mmol) and
triethylamine (417 .mu.L, 1.5 mmol). The resulting mixture was
stirred in dark from 0.degree. C. to RT for 16 h. The mixture was
then partitioned between ethyl acetate and brine. The organic layer
was dried over Na.sub.2SO.sub.4, filtered and concentrated in
vacuo. The residue was purified by column chromatography on silica
gel eluting with methanol and dichloromethane to afford the desired
product,
3-(4-amino-1-((8-methyl-1-oxo-2-o-tolyl-1,2-dihydroisoquinolin-3-yl)methy-
l)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-5-fluorophenyl diethyl
phosphate 4302 (200 mg, 62% yield) as an off-white solid.
3-(4-Amino-1-((8-methyl-1-oxo-2-o-tolyl-1,2-dihydroisoquinolin-3-yl)methy-
l)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-5-fluorophenyl diethyl
phosphate 4302 (170 mg, 0.26 mmol) was dissolved in anhydrous
CH.sub.3CN (5 mL) and cooled to 0.degree. C. under an argon
atmosphere. TMSBr (0.34 mL, 2.64 mmol) was slowly added via a
syringe and the resulting mixture was stirred from 0.degree. C. to
RT for 16 h. LC-MS showed small amount of staRTing material left,
additional amount of TMSBr (0.1 mL) was added and stirred at RT for
5 h. LC-MS showed the complete conversion. The mixture was
concentrated in vacuo, and the residue was dissolved in Et.sub.2O
(10 mL) and H.sub.2O (0.5 mL) and stirred for 30 min. The mixture
was concentrated in vacuo to affords the desired product,
3-(4-amino-1-((8-methyl-1-oxo-2-o-tolyl-1,2-dihydroisoquinolin-3-yl)methy-
l)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-5-fluorophenyl dihydrogen
phosphate 4303 (140 mg, 91% yield).
Example 9: Synthesis of
3-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-8-methyl-2-o--
tolylisoquinolin-1(2H)-one (compound 1611).
##STR00323## ##STR00324##
A mixture of 2,6-dimethylbenzoic acid (compound 4401) (60 g, 400
mmol) and oxalyl chloride (101 g, 800 mmol) in CH.sub.2Cl.sub.2
(400 mL) was stirred at room temperature for 2 h. The mixture was
concentrated in vacuo to afford the desired product,
2,6-dimethylbenzoyl chloride (compound 4402) (64 g, 95% yield) as a
yellow oil. The material obtained was used directly in the next
step without purification.
A mixture of o-toluidine (45 g, 420 mmol) and triethylamine (71 g,
700 mmol) in CH.sub.2Cl.sub.2 (300 mL) was stirred at room
temperature for 10 min. To this mixture, 2,6-dimethylbenzoyl
chloride (compound 4402) (64 g, 400 mmol) was added dropwise and
the resulting mixture was stirred at room temperature for 30 min.
The reaction mixture was poured into water (300 mL), extracted with
CH.sub.2Cl.sub.2 (3.times.200 mL), dried over anhydrous
Na.sub.2SO.sub.4 and filtered. The filtrate was concentrated in
vacuo to afford the crude product. The crude product was suspended
in isopropyl ether (300 mL), stirred at reflux for 30 min and then
was cooled to 0-5.degree. C. The solid was collected by filtration
and further dried in vacuo to afford the desired product,
2,6-dimethyl-N-o-tolylbenzamide (compound 4403) (81 g, 80% yield)
as a yellow solid.
To a stirred solution of 2,6-dimethyl-N-o-tolylbenzamide (compound
4403) (23.9 g, 0.1 mol, 1 eq) and HMPA (17.9 g, 0.1 mol, 1 eq) in
anhydrous THF (250 mL) at -78.degree. C. under an argon atmosphere,
n-butyllithium (100 mL, 2.5 M, 0.25 mol, 2.5 eq) was carefully
added over 1 h and the reaction temperature was kept below
-60.degree. C. during the addition. The resulting mixture was
stirred at -78.degree. C. for 1 h, and then diethyl oxalate (17.6
g, 0.12 mol, 1.2 eq) was quickly added (the reaction temperature
rose to -20.degree. C. upon addition). The mixture was stirred at
-50.degree. C. for 10 min, and then quenched with water (100 mL).
The inorganic salt was removed by filtration, and the filtrate was
extracted with ethyl acetate (2.times.100 mL). The combined organic
layer was washed with brine (100 mL), dried over MgSO.sub.4 and
filtered. The filtrate was concentrated in vacuo to afford the
crude product as a semi-solid oil. The crude product was slurried
in isopropyl ether (100 mL) at room temperature for 10 min. The
solid was collected by filtration and further dried in vacuo to
afford the desired product, ethyl
3-(3-methyl-2-(o-tolylcarbamoyl)phenyl)-2-oxopropanoate (compound
4404) (16.1 g, 47.4% yield) as a white solid.
3-(3-Methyl-2-(o-tolylcarbamoyl)phenyl)-2-oxopropanoate (compound
4404) (11.0 g, 32.4 mmol, 1 eq) was dissolved in HCl/MeOH (10 M,
100 mL, 10 mL/1 g of 4404) and stirred at reflux for 1 h. The
reaction mixture was concentrated in vacuo, and the residue was
slurried in ethyl acetate (10 mL) at room temperature for 30 min.
The solid was collected by filtration and further dried in vacuo to
afford the desired product, ethyl
8-methyl-1-oxo-2-o-tolyl-1,2-dihydroisoquinoline-3-carboxylate
(compound 4405) (7.52 g, 72.5% yield) as a white solid.
To a stirred solution of lithium aluminum hydride (8.28 g, 218 mol)
in anhydrous THF (500 mL) at -78.degree. C. under a nitrogen
atmosphere, ethyl
8-methyl-1-oxo-2-o-tolyl-1,2-dihydroisoquinoline-3-carboxylate
(compound 4405) (28 g, 87 mmol) was slowly added over a 10 min
period of time. The resulting mixture was allowed to warm to
-30.degree. C., stirred for 30 min and analysis by thin layer
chromatography showed completion of the reaction. Then the mixture
was cooled to -78.degree. C., and water (50 mL) was slowly added.
The mixture was allowed to warm to room temperature, filtered
through silica gel (10 g), and the filtrate was concentrated in
vacuo. The crude product was poured into H.sub.2O (200 mL) and
extracted with ethyl acetate (3.times.200 mL). The combined organic
layer was washed with brine (50 mL), dried over Na.sub.2SO.sub.4
and filtered. The filtrate was concentrated in vacuo. The crude
product was suspended in ethyl acetate (30 mL) and stirred for 10
min. The solid was collected by filtration and further dried in
vacuo to afford the desired product,
3-(hydroxymethyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one (compound
4406) (22 g, 92% yield) as a white solid.
Phosphorus tribromide (25.6 g, 95 mmol) was slowly added to a
stirred solution of DMF (11.5 g, 158 mol) in acetonitrile (200 mL)
at 0.degree. C., and the resulting mixture was stirred at 0.degree.
C. for 30 min.
3-(Hydroxymethyl)-8-methyl-2-o-tolylisoquinolin-1-(2H)-one
(compound 4406) (22 g, 78.8 mmol) was slowly added. Then the
reaction mixture was allowed to warm to room temperature and
stirred for 30 min. saturated aqueous NaHCO.sub.3 solution (50 mL)
was slowly added and extracted with ethyl acetate (3.times.200 mL).
The combined organic layer was washed with brine, dried over
Na.sub.2SO.sub.4 and filtered. The filtrate was concentrated in
vacuo. The crude product was suspended in isopropyl ether (50 mL)
and then stirred for 10 min. The precipitate was collected by
filtration and further dried in vacuo to afford the desired
product, 3-(bromomethyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one
(compound 4407) (21 g, 80% yield) as a white solid.
3-Iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (10.8 g, 41.4 mmol) and
potassium tert-butoxide (4.4 g, 40 mmol) were dissolved in
anhydrous DMF (150 mL) and stirred at room temperature for 30 min.
3-(Bromomethyl)-8-methyl-2-o-tolylisoquinolin-1(2H)-one (compound
4407) (13.7 g, 40 mmol) was added. The resulting mixture was
stirred at room temperature for 30 min, poured into ice water (300
mL) and then extracted with ethyl acetate (3.times.200 mL). The
combined organic layer was washed with brine (50 mL), dried over
Na.sub.2SO.sub.4 and filtered. The filtrate was concentrated to
about 100 ml in vacuo, the precipitate was collected by filtration
to afford the first batch of desired product,
3-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-8-methyl-2-o--
tolylisoquinolin-1(2H)-one (compound 1611) (12 g, 60% yield) as a
white solid. The filtrate was concentrated in vacuo and the residue
was purified by flash column chromatography on silica gel (2-20%
MeOH/DCM) to afford the second batch of desired product,
3-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-8-methyl-2-o--
tolylisoquinolin-1(2H)-one (compound 1611) (6 g, 30% yield) as a
white solid.
Example 10: Synthesis of
3-((4-amino-3-(fluoromethyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-8-m-
ethyl-2-o-tolylisoquinolin-1(2H)-one (compound 4504).
##STR00325## ##STR00326##
To a stirred mixture of
3-((4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-8-methyl-2-o--
tolylisoquinolin-1(2H)-one (compound 1611) (1.50 g, 2.87 mmol) and
tetrakis(triphenylphosphine)palladium (166 mg, 0.14 mmol) in
anhydrous DMF (15 mL) under an argon atmosphere, tributyl vinyl tin
(1.26 mL, 4.31 mmol) was added and the resulting mixture was
stirred at 80.degree. C. for 3 h. The reaction mixture was allowed
to cool to room temperature, and then partitioned between water and
ethyl acetate. The organic layer was washed with brine, dried over
Na.sub.2SO.sub.4 and filtered. The filtrate was concentrated in
vacuo and the residue was triturated with a minimal amount of
anhydrous ethyl ether and filtered to afford the desired product,
3-((4-amino-3-vinyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-8-methyl-2-o-
-tolylisoquinolin-1(2H)-one (compound 4501) (853 mg, 70% yield) as
an off-white solid.
To a stirred solution of
3-((4-amino-3-vinyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-8-methyl-2-o-
-tolylisoquinolin-1(2H)-one (compound 4501) (853 mg, 2.0 mmol) in
1,4-dioxane-H.sub.2O (3:1, 30 mL) under an argon atmosphere, osmium
tetroxide (2.5 wt % in t-BuOH, 252 .mu.L, 0.020 mmol) was added and
the resulting mixture was stirred at RT for 30 min. To this
mixture, sodium periodate (863 mg, 4.0 mmol) was added and the
resulting mixture was stirred for 3 h. The reaction mixture
partitioned between water and ethyl acetate. The organic layer was
washed with brine, dried over Na.sub.2SO.sub.4 and filtered. The
filtrate was concentrated in vacuo to afford the desired product,
4-amino-1-((8-methyl-1-oxo-2-o-tolyl-1,2-dihydroisoquinolin-3-yl)methyl)--
1H-pyrazolo[3,4-d]pyrimidine-3-carbaldehyde as a tan/brown solid
(compound 4502) (716 mg, 84% yield).
To a stirred mixture of
4-amino-1-((8-methyl-1-oxo-2-o-tolyl-1,2-dihydroisoquinolin-3-yl)methyl)--
1H-pyrazolo[3,4-d]pyrimidine-3-carbaldehyde as a tan/brown solid
(compound 4502) (841 mg, 1.98 mmol) in anhydrous MeOH (35 mL) at
0.degree. C. under an argon atmosphere, NaBH.sub.4 (89 mg, 2.38
mmol) was added in portions. The mixture was stirred from 0.degree.
C. to RT for 2 h, and then was partitioned between water and ethyl
acetate. The organic layer was washed with brine, dried over
Na.sub.2SO.sub.4 and filtered. The filtrate was concentrated in
vacuo to afford the desired product,
3-((4-amino-3-(hydroxymethyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-8--
methyl-2-o-tolylisoquinolin-1(2H)-one (compound 4503) (626 mg, 74%
yield) as dark brown solid.
To a stirred suspension of
3-((4-amino-3-(hydroxymethyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-8--
methyl-2-o-tolylisoquinolin-1(2H)-one (compound 4503) (50 mg, 0.12
mmol) in anhydrous DCM (2 mL) at 0.degree. C. under an argon
atmosphere, diethylaminosulfur trifluoride (DAST, 77 .mu.L, 0.59
mmol) was slowly added and the resulting mixture was stirred from
0.degree. C. to room temperature for 5 h. The reaction was quenched
with water and extracted with ethyl acetate. The combined organic
layer was washed with brine, dried over Na.sub.2SO.sub.4 and
filtered. The filtrate was concentrated in vacuo and the residue
was purified by prep-TLC plate (7% MeOH/DCM) to afford the desired
product,
3-((4-amino-3-(fluoromethyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-8-m-
ethyl-2-o-tolylisoquinolin-1(2H)-one (compound 4504) (10.3 mg, 20%
yield) as a white solid.
Example 11: Synthesis of
4-amino-1-((8-methyl-1-oxo-2-o-tolyl-1,2-dihydroisoquinolin-3-yl)methyl)--
1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide (compound 4602).
##STR00327##
To a stirred solution of
4-amino-1-((8-methyl-1-oxo-2-o-tolyl-1,2-dihydroisoquinolin-3-yl)methyl)--
1H-pyrazolo[3,4-d]pyrimidine-3-carbaldehyde (compound 4502) (400
mg, 0.94 mmol) in t-BuOH (1.8 mL), a solution of NaH.sub.2PO.sub.4
(3.90 g, 28.27 mmol) in water (4.8 mL), methyl-2-butene (1.0 mL)
and (dropwise) a solution of NaClO.sub.2 (767 mg, 6.78 mmol) in
water (4.8 mL) were added sequentially. The mixture was stirred at
RT for 3 h under an argon atmosphere. The pale yellow solution was
acidified with aqueous HCl solution (2 M, 4 mL) to PH=2 and
extracted with ethyl acetate. The combined organic layer was washed
with brine, dried over anhydrous Na.sub.2SO.sub.4 and filtered. The
filtrate was concentrated in vacuo, and the residue was triturated
with anhydrous ethyl ether and ethyl acetate. The solid was
collected by filtration to afford the desired product,
4-amino-1-((8-methyl-1-oxo-2-o-tolyl-1,2-dihydroisoquinolin-3-yl-
)methyl)-1H-pyrazolo[3,4-d]pyrimidine-3-carboxylic acid (compound
4601) (200 mg, 47% yield) as a yellow solid.
To a stirred solution of
4-amino-1-((8-methyl-1-oxo-2-o-tolyl-1,2-dihydroisoquinolin-3-yl)methyl)--
1H-pyrazolo[3,4-d]pyrimidine-3-carboxylic acid (compound 4601) (150
mg, 0.34 mmol) in anhydrous DCM (10 mL), oxalyl chloride (2.0 M in
DCM, 0.22 mL) was slowly added followed by a catalytic amount of
anhydrous DMF (1 drop). The resulting mixture was stirred at room
temperature for 30 min and then concentrated in vacuo. The residue
was re-dissolved in DCM (6 mL) and an excess amount of ammonium
hydroxide was added (0.35 mL). The mixture was stirred at room
temperature for 2 h, and then partitioned between ethyl acetate and
water. The organic layer was washed with brine, dried over
anhydrous Na.sub.2SO.sub.4 and filtered. The filtrate was
concentrated in vacuo, and the residue was purified by flash column
chromatography on silica gel (eluting with 5% MeOH/DCM) to afford
the desired product,
4-amino-1-((8-methyl-1-oxo-2-o-tolyl-1,2-dihydroisoquinolin-3-yl)methyl)--
1H-pyrazolo[3,4-d]pyrimidine-3-carboxamide (compound 4602) (22 mg,
15% yield) as a white solid.
Example 12: Synthesis of
(S)-3-(1-(9H-purin-6-ylamino)ethyl)-8-methyl-2-phenylisoquinolin-1(2H)-on-
e (compound 4704) (Method A)
##STR00328##
To a stirred mixture of (S)-2-(tert-butoxycarbonylamino)propanoic
acid (compound 4701) (189.1 g, 1 mol, 1 eq), triethylamine (404.8
g, 4 mol, 4 eq) and HOBt (135 g, 1.0 mol, 1 eq) in anhydrous
dichloromethane (1.8 L) at 0.degree. C., EDCI (384.3 g, 2 mol, 2
eq) was added in portions over 30 min. The resulting mixture was
stirred at RT for 30 min, and then N,O-dimethylhydroxylamine
hydrochloride (107.3 g, 1.1 mol, 1.1 eq) was added. The reaction
mixture was stirred at RT for 20 h, and then quenched with water (1
L). The organic layer was washed with water (2.times.1 L) and brine
(500 mL), dried over anhydrous MgSO.sub.4 and filtered. The
filtrate was concentrated in vacuo. The crude product was slurried
in petroleum ether (1 L) and stirred at RT for 10 min. The solid
was collected by filtration and further dried in vacuo to afford
the desired product, (S)-tert-butyl
1-(methoxy(methyl)amino)-1-oxopropan-2-ylcarbamate (compound 4702)
(218 g, 93.9% yield) as a white solid.
To a stirred mixture of 2,6-dimethyl-N-phenylbenzamide (compound
4403, which may be synthesized as described in Example 9) (30 g,
0.13 mol, 1 eq) and HMPA (26 g, 0.16 mol, 1.2 eq) in anhydrous THF
(300 mL) at -78.degree. C. under an argon atmosphere,
n-butyllithium (2.5 M, 100 mL, 0.25 mol, 2.5 eq) was carefully
added (dropwise) over a 1 h and the reaction temperature was kept
below -60.degree. C. during the addition. The resulting mixture was
stirred at -78.degree. C. for 1 h. To this mixture, tert-butyl
1-(methoxy(methyl)amino)-1-oxopropan-2-yl carbamate (compound 4702)
(40 g, 0.173 mol, 1.3 eq) was quickly added (the reaction
temperature rose to -50.degree. C. upon addition). The mixture was
stirred at -50.degree. C. for 10 min, quenched with water (300 mL)
and extracted with ethyl acetate (2.times.100 mL). The combined
organic layer was washed with water (500 mL.times.2) and brine (50
mL), dried over anhydrous MgSO.sub.4 and filtered. The filtrate was
concentrated in vacuo to give the crude product as a semi-solid
oil. The crude product was slurried in EA and stirred for 10 min.
The white solid was removed by filtration. The filtrate was
concentrated in vacuo, and the residue was stirred in a mixture of
ethyl acetate (30 mL) and isopropyl alcohol (200 mL) at RT for 10
min. The solid was collected by filtration and further dried in
vacuo to afford the desired product,
tert-butyl4-(3-methyl-2-(phenylcarbamoyl)phenyl)-3-oxobutan-2-yl
carbamate (compound 4703) (9.23 g, 17.5% yield) as a white
solid.
Tert-Butyl-4-(3-methyl-2-(phenylcarbamoyl)phenyl)-3-oxobutan-2-yl
carbamate (compound 4703) (9.23 g, 23 mmol) was dissolved in
HCl/MeOH (100 mL) and stirred at reflux for 30 min. The mixture was
allowed to cool to RT, concentrated in vacuo, and then saturated
Na.sub.2CO.sub.3 solution was added to adjust the PH to 7-8. The
solid was collected by filtration and further dried in vacuo to
afford the desired product,
3-(1-aminoethyl)-8-methyl-2-phenylisoquinolin-1(2H)-one (compound
4704) (5.8 g, 90% yield, S:R isomers=7:1) as a white solid.
##STR00329##
Resolution of Isomers to Increase the Enantiomeric Purity:
3-(1-Aminoethyl)-8-methyl-2-phenylisoquinolin-1(2H)-one (compound
4704) (where the ratio of isomers is S:R=7:1) (5 g, 18 mmol) was
dissolved in MeOH (100 mL), (D)-tartaric-acid (2.7 g, 18 mmol) was
added. The mixture was stirred at RT for 30 min and the solid was
precipitated. The resulting mixture was stirred at reflux for 1 h,
and then stirred at RT for 16 h. The solid was collected by
filtration and rinsed with methanol (10 mL). The solid was then
dissolved in H.sub.2O (15 mL) and saturated NaHCO.sub.3 (5 mL) was
added to adjust the PH to 8. The solid was collected by filtration,
rinsed with water (5 mL), and then dried in vacuo to afford the
enantiomerically enriched product (compound 4704) (2.7 g, 58%
yield,) where the ratio of isomers, S:R>41:1. This an
enantiomeric purity of greater than about 97.6% of the
(S)-enantiomer. The ratio of two enantiomers was confirmed by
coupling with (R)-(-)-alpha-methoxyphenylacetic acid and detection
of the resultant diastereomers by Nuclear Magnetic Resonance
Spectroscopy.
Example 13: Synthesis of
(S)-3-(1-aminoethyl)-8-methyl-2-phenylisoquinolin-1(2H)-one (Method
B) (compound 4704.
##STR00330##
Thionyl chloride (320.8 g, 2.7 mol, 1.2 eq) was added dropwise to
stirred anhydrous MeOH (2 L) at 0.degree. C. over 50 min and the
reaction temperature was kept below 25.degree. C. during the
addition. The mixture was allowed to warm to room temperature and
then (S)-2-aminopropanoic acid (compound 4801) (200 g, 2.24 mol, 1
eq) was added. The resulting mixture was stirred at room
temperature for 20 h, and concentrated in vacuo to afford the
desired product, (S)-methyl 2-aminopropanoate hydrochloride
(compound 4802) as a white solid.
To a stirred solution of above obtained (S)-methyl
2-aminopropanoate hydrochloride (compound 4802) in water (1.6 L) at
room temperature, NaHCO.sub.3 (566.2 g, 6.741 mol, 3 eq) and a
solution of di-tert-butyl dicarbonate (490.4 g, 2.247 g, 1 eq) in
THF (1.6 L) were added sequentially. The resulting mixture was
stirred at room temperature for 20 h. The inorganic salt was
removed by filtration, and the filtrate was extracted with ethyl
acetate (2.times.500 mL). The combined organic layer was washed
with brine (500 mL), dried over anhydrous MgSO.sub.4 and filtered.
The filtrate was concentrated in vacuo to afford the desired
product, (S)-methyl 2-(tert-butoxycarbonylamino)propanoate
(compound 4803) (448 g, 98.2% yield) as a colorless crystal.
To a stirred solution of 2,6-dimethyl-N-phenylbenzamide (compound
4403), which may be synthesized as described in Example 9) (30 g,
0.13 mol, 1 eq) and HMPA (26 g, 0.16 mol, 1.2 eq) in anhydrous THF
(300 mL) at -78.degree. C. under an argon atmosphere,
n-butyllithium (100 mL, 2.5 M, 0.25 mol, 2.5 eq) was added
carefully over 1 h and the reaction temperature was kept below
-60.degree. C. during addition. The resulting mixture was stirred
at -78.degree. C. for 1 h, and then (S)-methyl
2-(tert-butoxycarbonylamino)-propanoate (compound 4803) (35 g,
0.173 mol, 1.3 eq) was quickly added (the reaction temperature rose
to -50.degree. C. during addition). The mixture was stirred at
-50.degree. C. for 10 min, quenched with water (300 mL) and
extracted with ethyl acetate (2.times.100 mL). The organic layer
was washed with water (500 mL.times.2), dried over anhydrous
MgSO.sub.4 and filtered. The filtrate was concentrated in vacuo to
afford the crude product as a semi-solid oil. The crude product was
slurried in ethyl acetate (500 mL) and stirred for 10 min. The
solid was removed by filtration, and the filtrate was concentrated
in vacuo. The oil residue was stirred in a mixture of ethyl acetate
(30 mL) and isopropyl alcohol (200 mL) at room temperature for 10
min. The solid was collected by filtration and further dried in
vacuo to afford the desired product,
tert-butyl4-(3-methyl-2-(phenylcarbamoyl)phenyl)-3-oxobutan-2-ylcarbamate
(compound 4703) (4.61 g, 9% yield) as a white solid.
Tert-Butyl
4-(3-methyl-2-(phenylcarbamoyl)phenyl)-3-oxobutan-2-ylcarbamate
(compound 4703) (4.61 g, 0.012 mol) was dissolved in HCl/MeOH (50
mL) and stirred at reflux for 30 min. The mixture was concentrated
in vacuo and then saturated Na.sub.2CO.sub.3 solution was added to
adjust PH to about 7-8. The resulting solid was collected by
filtration and further dried in vacuo to afford the desired
product, 3-(1-aminoethyl)-8-methyl-2-phenylisoquinolin-1(2H)-one
(compound 4704) (2.9 g, 90% yield, where the ratio of isomers is
S:R=5:1) as a white solid.
Example 14a: Synthesis of
(S)-3-(1-(9H-purin-6-ylamino)ethyl)-8-methyl-2-phenylisoquinolin-1(2H)-on-
e (9) (compound 4902)
##STR00331##
3-(1-Aminoethyl)-8-methyl-2-phenylisoquinolin-1(2H)-one (compound
4704) (200 mg, 0.72 mmol),
6-chloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purine (344 mg, 1.44 mmol)
and DIPEA (279 mg, 2.16 mmol) were dissolved in n-BuOH (20 ml), and
the resulting mixture was stirred at reflux for 16 h. The reaction
mixture was concentrated in vacuo and purified by flash column
chromatography on silica gel (eluting with 30% to 50% Hex/EA) to
afford the desired product,
8-methyl-2-phenyl-3-((1S)-1-(9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-ylam-
ino)ethyl)isoquinolin-1(2H)-one (compound 4901) (207 mg, 60% yield)
as a white solid.
8-Methyl-2-phenyl-3-((1S)-1-(9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-ylam-
ino)ethyl)-isoquinolin-1(2H)-one (compound 4901) (200 mg, 0.42
mmol) was dissolved in HCl/EtOH (3 M, 5 mL) and the resulting
mixture was stirred at room temperature for 1 h. The reaction
mixture was quenched with saturated NaHCO.sub.3 aqueous solution
and the PH was adjusted to about 7-8. The mixture was extracted
with CH.sub.2Cl.sub.2 (50 mL.times.3), dried over anhydrous
Na.sub.2SO.sub.4 and filtered. The filtrate was concentrated in
vacuo, and the residue was recrystallized from ethyl acetate and
hexanes (1:1). The solid was collected by filtration and dried in
vacuo to afford the desired product,
(S)-3-(1-(9H-purin-6-ylamino)ethyl)-8-methyl-2-phenylisoquinolin-1(2H)-on-
e (compound 4902) (150 mg, 90% yield) as a white solid.
Example 14b: Synthesis of
(S)-3-(1-(9H-purin-6-ylamino)ethyl)-8-chloro-2-phenylisoquinolin-1(2H)-on-
e (9) (compound 4904)
##STR00332##
The compound of Formula 4904 was synthesized using the synthetic
transformations as described in Examples 13 and 14a, but
2-chloro-6-methyl benzoic acid (compound 4903) was used instead of
2,6,dimethyl benzoic acid (compound 4403).
Example 15a: Synthesis of
(S)-3-(1-(9H-purin-6-ylamino)ethyl)-2-cyclopropyl-8-methylisoquinolin-1(2-
H)-one (compound 5005).
##STR00333##
A mixture of cyclopropanamine (24 g, 420 mmol) and triethylamine
(71 g, 700 mmol) in CH.sub.2Cl.sub.2 (300 mL) was stirred at RT for
10 min. To this mixture, 2,6-dimethylbenzoyl chloride (compound
4402) (64 g, 400 mmol) was added dropwise, and the resulting
mixture was stirred at room temperature for 30 min. The reaction
mixture was poured into water (300 mL) and extracted with
CH.sub.2Cl.sub.2 (3.times.200 mL). The combined organic layer was
dried over anhydrous Na.sub.2SO.sub.4 and filtered. The filtrate
was concentrated in vacuo to afford the crude product. The crude
product was suspended in isopropyl ether (IPE) (300 mL), stirred at
reflux for 30 min and then was allowed to cool to 0-5.degree. C.
The precipitate was collected by filtration and further dried in
vacuo to afford the desired product,
N-cyclopropyl-2,6-dimethylbenzamide (compound 5001) (61 g, 80%
yield) as a yellow solid.
To a stirred solution of N-cyclopropyl-2,6-dimethylbenzamide
(compound 5001) (25 g, 0.13 mol, 1 eq) and HMPA (26 g, 0.16 mol,
1.2 eq) in anhydrous THF (300 mL) at -78.degree. C. under an argon
atmosphere, n-butyllithium (2.5M, 100 mL, 0.25 mol, 2.5 eq) was
added carefully over 1 h and the temperature was kept below
-60.degree. C. during addition. The resulting mixture was stirred
at -78.degree. C. for 1 h, and then tert-butyl
1-(methoxy(methyl)amino)-1-oxopropan-2-ylcarbamate (40 g, 0.173
mol, 1.3 eq) was quickly added (the reaction temperature rose to
-50.degree. C. during addition). The mixture was stirred at
-50.degree. C. for 10 min, quenched with water (300 mL) and
extracted with ethyl acetate (2.times.100 mL). The combined organic
layer was washed with water (500 mL.times.2) and brine (100 mL),
dried over anhydrous MgSO.sub.4 and filtered. The filtrate was
concentrated in vacuo to afford the desired product, tert-butyl
4-(2-(cyclopropylcarbamoyl)-3-methylphenyl)-3-oxobutan-2-ylcarbamate
(compound 5002) (32 g, 70% yield) as a yellow oil.
Tert-Butyl
4-(2-(cyclopropylcarbamoyl)-3-methylphenyl)-3-oxobutan-2-ylcarbamate
(compound 5002) (32 g, 88 mmol) was dissolved in HCl/MeOH (300 mL)
and stirred at room temperature for 16 h. The mixture was
concentrated in vacuo, and then saturated Na.sub.2CO.sub.3 aqueous
solution was added to adjust the pH to about 7-8. The resulting
solid was collected by filtration and further dried in vacuo to
afford the desired product,
3-(1-aminoethyl)-8-methyl-2-phenylisoquinolin-1(2H)-one (compound
5003) (17 g, 80% yield, S:R=7:1) as a white solid.
##STR00334##
To a stirred solution of
3-(1-aminoethyl)-2-cyclopropyl-8-methylisoquinolin-1(2H)-one
(S:R=7:1) (4.84 g, 20 mmol) (compound 5003) in MeOH (96.8 mL), (L)
tartaric-acid (3.0 g, 20 mmol) was added and the resulting mixture
was stirred at room temperature for 16 h. The precipitate was
collected by filtration and rinsed with MeOH (10 mL). The solid was
dissolved in H.sub.2O (15 mL) and statured NaHCO.sub.3 (5 mL) was
added to adjust the pH to about 8. The resulting solid was
collected by filtration, rinsed with water (5 mL), and dried in
vacuo to afford the desired product (compound 5003) (1.94 g. 40%
yield) as a single enantiomer (S configuration). The enantiomeric
purity was confirmed by coupling with
(R)-(-)-alpha-methoxyphenylacetic acid and performing Nuclear
Magnetic Resonance Spectroscopy on the resulting diastereomeric
mixture.
(S)-3-(1-Aminoethyl)-2-cyclopropyl-8-methylisoquinolin-1(2H)-one
(242 mg, 1 mmol) (compound 5003),
6-chloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purine (344 mg, 1.44 mmol)
and DIPEA (279 mg, 2.16 mmol) were dissolved in n-BuOH (20 mL), and
the resulting mixture was stirred at reflux for 16 h. The reaction
mixture was concentrated in vacuo and the residue was purified by
flash column chromatography on silica gel (eluting with 30% to 50%
Hex/EA) to afford the desired product,
2-cyclopropyl-8-methyl-3-((1S)-1-(9-(tetrahydro-2H-Pyran-2-yl)-9H-purin-6-
-ylamino)ethyl)isoquinolin-1(2H)-one (compound 5004) (288 mg, 65%
yield) as a white solid.
2-Cyclopropyl-8-methyl-3-((1S)-1-(9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-
-ylamino)ethyl)isoquinolin-1(2H)-one (compound 5004) (222 mg, 0.5
mmol) was dissolved in HCl/EtOH (3 M, 5 mL) and the resulting
mixture was stirred at room temperature for 1 h. The reaction
mixture was neutralized with saturated NaHCO.sub.3 solution to
pH=7-8, and then extracted with CH.sub.2Cl.sub.2 (50 mL.times.3).
The combined organic layer was washed with brine, dried over
anhydrous Na.sub.2SO.sub.4 and filtered. The filtrate was
concentrated in vacuo and the residue was recrystallized from ethyl
acetate and hexanes (1:1). The solid was collected by filtration
and dried in vacuo to afford the desired product,
(S)-3-(1-(9H-purin-6-ylamino)ethyl)-2-cyclopropyl-8-methylisoquinolin-1(2-
H)-one (compound 5005) (150 mg, 83% yield) as a white solid.
Example 15b. Synthesis of
(S)-3-(1-(9H-purin-6-ylamino)ethyl)-2-cyclopropyl-8-chloro-isoquinolin-1(-
2H)-one (compound 5011).
##STR00335##
The compound of Formula 5011 was synthesized using the synthetic
transformations as described in Example 15a, but 2-chloro-6-methyl
benzoyl chloride (compound 5006) was used instead of 2,6,dimethyl
benzoyl chloride (compound 4402).
Example 16: Synthesis of
(S)-3-(1-(2-amino-5-chloropyrimidin-4-ylamino)ethyl)-8-methyl-2-phenyliso-
quinolin-1(2H)-one (compound 5102).
##STR00336##
A mixture of
3-(1-aminoethyl)-8-methyl-2-phenylisoquinolin-1(2H)-one (compound
4704) (150 mg, 0.54 mmol), 2,4,5-trichloropyrimidine (119 mg, 0.65
mmol) and triethylamine (137 mg, 1.35 mmol) in n-BuOH (10 mL) was
stirred at reflux for 2 h. The mixture was allowed to cool to room
temperature and then concentrated in vacuo. The residue was
purified by flash column chromatography on silica gel
(MeOH:CH.sub.2Cl.sub.2=1:100) to afford the desired product,
(S)-3-(1-(2,5-dichloropyrimidin-4-ylamino)ethyl)-8-methyl-2-phenylisoquin-
olin-1(2H)-one (2H)-one (compound 5101) (170 mg, 74% yield) as a
white solid.
A mixture of
(S)-3-(1-(2,5-dichloropyrimidin-4-ylamino)ethyl)-8-methyl-2-phenylisoquin-
olin-1(2H)-one (compound 5101) (85 mg, 0.20 mmol) in ammonia water
(15 mL) in a sealed tube was stirred at 150.degree. C. for 16 h.
The solution was allowed to cool to room temperature and then
partitioned between water (30 mL) and ethyl acetate (3.times.30
mL). The combined organic layer was washed with brine (2.times.20
mL), dried over anhydrous Na.sub.2SO.sub.4 and filtered. The
filtrate was concentrated in vacuo to afford the desired product,
(S)-3-(1-(2-amino-5-chloropyrimidin-4-ylamino)ethyl)-8-methyl-2-phenyliso-
quinolin-1(2H)-one (compound 5102) (40 mg, 49.6% yield) as a white
solid.
Example 17: Synthesis of
(S)-3-(1-(2-fluoro-9H-purin-6-ylamino)ethyl)-8-methyl-2-phenylisoquinolin-
-1(2H)-one (compound 5204).
##STR00337##
To a stirred mixture of 6-chloro-2-fluoro-9H-purine (compound 5201)
(2.07 g, 12.0 mmol) and p-toluenesulfonic acid monohydrate (34 mg,
0.18 mmol) in ethyl acetate (50 mL) under an argon atmosphere,
3,4-dihydropyran (3.03 g, 36.0 mmol) was added and the resulting
mixture was stirred at reflux for 16 h. The reaction mixture was
concentrated in vacuo and the residue was purified by flash column
chromatography on silica gel (eluting with 10% Hex/EA) to afford
the desired product,
6-chloro-2-fluoro-9-(tetrahydro-2H-pyran-2-yl)-9H-purine (compound
5202) (1.82 g, 59% yield) as a white solid.
3-(1-Aminoethyl)-8-methyl-2-phenylisoquinolin-1(2H)-one (200 mg,
0.72 mol), 6-chloro-2-fluoro-9-(tetrahydro-2H-pyran-2-yl)-9H-purine
(compound 5202) (369 mg, 1.44 mmol) and DIPEA (279 mg, 2.16 mmol)
were dissolved in n-BuOH (20 mL) in a sealed tube, and the
resulting mixture was stirred at 120.degree. C. for 16 h. The
reaction mixture was concentrated in vacuo and the residue was
purified by flash column chromatography on silica gel (eluting with
30% to 50% Hex/EA) to afford the desired product,
3-(1-(2-fluoro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-ylamino)ethyl)-8-met-
hyl-2-phenylisoquinolin-1(2H)-one (compound 5203) (167 mg, 47%
yield) as a white solid.
3-(1-(2-Fluoro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-ylamino)ethyl)-8-m-
ethyl-2-phenylisoquinolin-1(2H)-one (compound 5203) (160 mg, 0.32
mmol) was dissolved in HCl/EtOH (3 M, 5 mL) and the resulting
mixture was stirred at room temperature for 1 h. The mixture was
neutralized with saturated NaHCO.sub.3 aqueous solution to pH=7-8,
and extracted with CH.sub.2Cl.sub.2 (50 mL.times.3). The combined
organic layer was washed with brine, dried over anhydrous
Na.sub.2SO.sub.4 and filtered. The filtrate was concentrated in
vacuo and the residue was recrystallized from ethyl acetate and
hexanes. The solid was collected by filtration and dried in vacuo
to afford the desired product,
3-(1-(2-fluoro-9H-purin-6-ylamino)ethyl)-8-methyl-2-phenylisoquinolin-1(2-
H)-one (compound 5204) (125 mg, 94% yield) as a white solid.
Example 18: Synthesis of
(S)-3-(1-(2-chloro-9H-purin-6-ylamino)ethyl)-8-methyl-2-phenylisoquinolin-
-1(2H)-one (compound 5304).
##STR00338##
To a stirred mixture of 2,6-dichloro-9H-purine (compound 5301)
(2.27 g, 12.0 mmol) and p-toluenesulfonic acid monohydrate (34 mg,
0.18 mmol) in ethyl acetate (50 mL) under an argon atmosphere,
3,4-dihydropyran (3.03 g, 36.0 mmol) was added and the resulting
mixture was stirred at reflux for 16 h. The reaction mixture was
concentrated in vacuo and the residue was purified by flash column
chromatography on silica gel (eluting with 10% Hex/EA) to afford
the desired product,
2,6-dichloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purine (compound 5302)
(2.04 g, 62% yield) as a white solid.
3-(1-Aminoethyl)-8-methyl-2-phenylisoquinolin-1(2H)-one (compound
4704) (200 mg, 0.72 mol),
2,6-dichloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purine (compound 5302)
(393 mg, 1.44 mmol) and DIPEA (279 mg, 2.16 mmol) were dissolved in
n-BuOH (20 mL) in a sealed tube, and the resulting mixture was
stirred at 120.degree. C. for 16 h. The reaction mixture was
concentrated in vacuo and the residue was purified by flash column
chromatography on silica gel (eluting with 30% to 50% Hex/EA) to
afford the desired product,
3-(1-(2-chloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-ylamino)ethyl)-8-m-
ethyl-2-phenylisoquinolin-1(2H)-one (compound 5303) (172 mg, 46%
yield) as a white solid.
3-(1-(2-Chloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-ylamino)ethyl)-8-m-
ethyl-2-phenylisoquinolin-1(2H)-one (compound 5303) (172 mg, 0.33
mmol) was dissolved in HCl/EtOH (3 M, 5 mL) and the resulting
mixture was stirred at room temperature for 1 h. The mixture was
neutralized with saturated NaHCO.sub.3 aqueous solution to pH=7-8,
and then extracted with CH.sub.2Cl.sub.2 (50 mL.times.3). The
combined organic layer was washed with brine, dried over
Na.sub.2SO.sub.4 and filtered. The filtrate was concentrated in
vacuo and recrystallized from ethyl acetate and hexanes. The solid
was collected by filtration and dried in vacuo to afford the
desired product,
3-(1-(2-chloro-9H-purin-6-ylamino)ethyl)-8-methyl-2-phenylisoquinolin-1(2-
H)-one (compound 5304) (128 mg, 90% yield) as a white solid.
Example 19: Synthesis of
(S)-3-(1-(2-amino-9H-purin-6-ylamino)ethyl)-8-methyl-2-phenylisoquinolin--
1(2H)-one (compound 5402).
##STR00339##
(S)-3-(1-Aminoethyl)-8-methyl-2-phenylisoquinolin-1(2H)-one
(compound 4704) (100 mg, 0.36 mmol), 2-Amino-6-chloropurine
(compound 5401) (60.9 mg, 0.36 mmol) and N,N-diisopropylethyl amine
(69 .mu.L, 0.40 mmol) were suspended in n-BuOH (4 mL) in a sealed
tube, and the resulting mixture was stirred at 100.degree. C. for
48 h and then at 120.degree. C. for 24 h. The mixture was allowed
to cool to room temperature and concentrate in vacuo to remove
n-BuOH. The residue was partitioned between ethyl acetate and
water. The organic layer was washed with brine, dried over
Na.sub.2SO.sub.4 and filtered. The filtrate was concentrated in
vacuo. The residue was triturated with anhydrous ethyl ether and
further purified by flash column chromatography on silica gel
(eluting with 0-8% MeOH/DCM) to afford the desired product,
(S)-3-(1-(2-amino-9H-purin-6-ylamino)ethyl)-8-methyl-2-phenylisoquinolin--
1(2H)-one as a off white/yellow solid (compound 5402), (28 mg,
20%).
Example 20: Synthesis of
(S)-4-(1-(8-methyl-1-oxo-2-phenyl-1,2-dihydroisoquinolin-3-yl)ethylamino)-
-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile (compound 5506).
##STR00340##
To a stirred mixture of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine
(compound 5501) (3.99 g, 26.0 mmol) in dry CH.sub.2Cl.sub.2 (150
mL) under an argon atmosphere, N-bromosuccinimide (6.02 g, 33.8
mmol) was added. The reaction mixture was stirred at room
temperature for 3 h, diluted with MeOH (30 mL), and then
concentrated in vacuo to yield a slight brown solid. The residue
was triturated with H.sub.2O (150 mL) and then recrystallized from
MeOH (120 mL). The solid was collected by filtration and dried in
vacuo to afford the desired product,
5-bromo-4-chloro-7H-pyrrolo[2,3-d]pyrimidine (compound 5502) (4.0
g, 66% yield) as a white solid.
To a stirred solution of
5-bromo-4-chloro-7H-pyrrolo[2,3-d]pyrimidine (compound 5502) (2.33
g, 10.0 mmol) in anhydrous THF (100 mL) at -78.degree. C. under an
argon atmosphere, a solution of n-BuLi (8.8 mL, 22.0 mmol) in THF
(50 mL) was added dropwise over 10 min. The reaction mixture was
stirred at -78.degree. C. for 1 h and then DMF (2.00 g, 11.0 mmol)
was added dropwise over 10 min. The reaction mixture was stirred at
-78.degree. C. for 30 min, and then was allowed to slowly warm to
room temperature and stirred at room temperature for 16 h. The
mixture was diluted with H.sub.2O (50 mL), and then concentrated in
vacuo to remove THF. The resulting slurry was treated with
saturated NH.sub.4Cl aqueous solution (50 mL), filtered, washed
with ethyl acetate (100 mL), and dried in vacuo to afford the
desired product,
4-chloro-7H-pyrrolo[2,3-d]pyrimidine-5-carbaldehyde (compound 5503)
(1.17 g, 65% yield) as a white solid.
To a stirred mixture of
4-chloro-7H-pyrrolo[2,3-d]pyrimidine-5-carbaldehyde (compound 5503)
(1.17 g, 6.47 mmol) in EtOH (25 mL), hydroxylamine hydrochloride
solid (0.54 g, 7.77 mmol) and a solution of NaOH (0.311 g, 7.77
mmol) in H.sub.2O (4 mL) were added sequentially. The reaction
mixture was stirred at room temperature for 30 min and diluted with
a sufficient amount of EtOH (30 mL) and stirring was continued for
30 min. The solid was collected by filtration, rinsed with H.sub.2O
(100 mL) and dried in vacuo to afford the desired product,
4-chloro-7H-pyrrolo[2,3-d]pyrimidine-5-carbaldehyde oxime (compound
5504) (0.89 g, 70% yield) as a mixture of isomers.
To a stirred mixture of
4-chloro-7H-pyrrolo[2,3-d]pyrimidine-5-carbaldehyde oxime (compound
5504) (865 mg, 4.40 mmol) in CH.sub.2Cl.sub.2 (20 mL), SOCl.sub.2
(3.1 mL, 43.7 mmol) was added and the resulting mixture was stirred
at room temperature for 16 h. The reaction mixture was concentrated
in vacuo. The residue was treated with ethyl acetate (20 mL),
H.sub.2O (20 mL) and then saturated NaHCO.sub.3 aqueous solution
(50 mL) to adjust pH to about 3-4. The mixture was stirred at room
temperature for 15 min and the solid was collected by filtration.
The filtrate was extracted with ethyl acetate (80 mL.times.3),
dried over Na.sub.2SO.sub.4 and filtered. The filtrate was
concentrated in vacuo to afford the second batch of product. The
combined solid was recrystallized from ethyl acetate and hexanes
(1:1, 20 mL). The solid was collected by filtration and dried in
vacuo to afford the desired product,
4-chloro-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile (compound 5505)
(763 mg, 97% yield).
(S)-3-(1-Aminoethyl)-8-methyl-2-phenylisoquinolin-1(2H)-one
(compound 4704) (208 mg, 0.75 mol),
4-chloro-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile (compound 5505)
(160 mg, 0.90 mmol) and Et.sub.3N (228 mg, 2.25 mmol) were
dissolved in n-BuOH (20 mL) in a sealed tube, and the resulting
mixture stirred at 150.degree. C. for 16 h. The reaction mixture
was concentrated in vacuo, and the residue was purified by flash
column chromatography on silica gel (eluting with 50% Hex/EA) to
afford the desired product,
(S)-4-(1-(8-methyl-1-oxo-2-phenyl-1,2-dihydroisoquinolin-3-yl)ethylamino)-
-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile (compound 5506) (90 mg,
28% yield) as a white solid.
Example 21: IC50 Values for Selected Compounds.
TABLE-US-00003 TABLE 3 In Vitro IC.sub.50 data for selected
compounds. + (greater than ++ (less than +++ (less than ++++ (less
than 10 microMolar) 10 microMolar) 1 microMolar 100 nM) IC50(nM)
Compound No. Compound No. Compound No. Compound No. PI3K .delta.
197, 199, 241, 259, 1, 5, 22, 27, 38, 39, 4, 14, 15, 17, 18, 21, 2,
3, 6, 7, 8, 9, 10, 11, 261, 263, 280, 282, 40, 41, 46, 92, 117, 26,
29, 31, 32, 34, 35, 12, 13, 16, 19, 20, 23, 283, 314, 315, 318,
118, 120, 129, 132, 36, 42, 43, 44, 45, 47, 24, 25, 28, 30, 33, 37,
321, 322 164, 165, 172, 188, 49, 57, 69, 71, 85, 87, 48, 50, 51,
52, 53, 54, 186, 193, 194, 195, 94, 106, 107, 143, 55, 56, 58, 59,
60, 61, 217, 242, 246, 281, 175, 179, 181, 182, 62, 63, 64, 65, 66,
67, 284, 305, 317, 325 183, 187, 189, 192, 68, 70, 72, 73, 74, 75,
225, 226, 228, 235, 76, 77, 78, 79, 80, 81, 236, 239, 248, 250, 82,
83, 84, 86, 88, 89, 258, 269, 274, 275, 90, 91, 93, 95, 96, 97,
285, 286, 297, 298, 98, 99, 100, 101, 102, 299, 300, 307, 309, 103,
104, 105, 108, 313, 319, 109, 110, 111, 112, 113, 114, 115, 119,
123, 124, 125, 126, 128, 134, 135, 136, 137, 138, 139, 141, 142,
144, 145, 146, 147, 148, 149, 150, 151. 152, 153, 154, 155, 156,
157, 158, 159, 160, 161, 162, 166, 167, 168, 169, 170, 171, 173,
174, 176, 177, 178, 180, 185, 188, 190, 191, 196, 198, 200, 201,
202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214,
215, 216, 218, 219, 220, 221, 222, 223, 224, 227, 229, 230, 231,
232, 233, 234, 237, 238, 240, 243, 244, 245, 247, 249, 251, 252,
253, 254, 255, 256, 257, 260, 262, 264, 265, 266, 267, 268, 270,
271, 272, 273, 276, 277, 278, 279, 287, 288, 289, 290, 291, 292,
293, 294, 295, 296, 301, 302, 303, 306, 308, 310, 311, 312, 316,
320, 323, 324 PI3K .gamma. 1, 4, 5, 18, 38, 43, 60, 17, 34, 35, 37,
38, 40, 2, 8, 9, 10, 11, 14, 15, 3, 6, 7, 12, 13, 16, 19, 69, 169,
172, 192, 42, 57, 61, 65, 91, 92, 20, 22, 27, 28, 39, 41, 21, 23,
24, 25, 26, 29, 193, 194, 199, 227, 94, 105, 107, 164, 46, 47, 49,
51, 55, 58, 30, 31, 33, 36, 44, 45, 228, 233, 259, 263, 170, 175,
179, 181, 66, 70, 71, 73, 76, 78, 48, 50, 52, 53, 54, 56, 280, 281,
282, 283, 183, 184, 186, 187, 80, 93, 98, 99, 100, 59, 62, 63, 64,
67, 68, 314, 315, 317, 318, 189, 195, 197, 219, 103, 104, 106, 108,
72, 74, 75, 77, 79, 81, 321, 322, 325 221, 224, 232, 239, 109, 161,
162, 163, 82, 83, 84, 86, 87, 88, 241, 242, 246, 248, 165, 166,
180, 188, 89, 90, 95, 96, 97, 258, 261, 274, 284, 202, 206, 209,
212, 101, 102, 142, 145, 285, 294, 299, 303, 214, 216, 218, 220,
146, 147, 148, 149, 305, 307, 309, 312, 222, 229, 234, 236, 150,
151, 152, 160, 313, 319 238, 250, 267, 268, 167, 168, 171, 173,
269, 271, 275, 279, 174, 176, 177, 178. 286, 293, 298, 300, 182,
185, 190, 191, 301, 308, 316 196, 198, 200, 201, 203, 204, 205,
207, 208, 210, 211, 213, 215, 223, 230, 231, 235, 237, 240, 243,
244, 245, 247, 249, 251, 252, 253, 254, 255, 256, 257, 260, 262,
264, 265, 266, 270, 272, 273, 276, 277, 278, 287, 288, 289, 290,
291, 292, 295, 296, 302, 304, 306, 310, 311, 320, 323, 324 PI3K
.alpha. 6, 8, 9, 10, 11, 12, 13, 3, 7, 63, 66, 84, 86, 53, 95, 101,
102, 145, 142, 148, 150, 153, 14, 15, 16, 17, 18, 19, 89, 90, 97,
108, 113, 147, 149, 151, 177, 154, 155, 156, 157, 20, 21, 22, 23,
24, 25, 115, 152, 168, 171, 208, 257, 260, 262, 158, 159, 176, 201,
26, 27, 28, 29, 30, 31, 173, 185, 190, 198, 264, 270, 272, 276, 252
32, 33, 34, 35, 36, 37, 203, 204, 205, 206, 277, 278, 287, 288, 39,
40, 41, 42, 43, 44, 207, 209, 210, 213, 289, 320, 323 45, 46, 47,
48, 49, 50, 223, 235, 237, 240, 51, 52, 54, 55, 56, 57, 243, 244,
245, 251, 58, 59, 60, 61, 62, 64, 253, 254, 255, 256, 65, 67, 68,
69, 70, 71, 269, 273, 279, 291, 72, 73, 74, 79, 80, 81, 292, 295,
296 82, 83, 85, 87, 88, 91, 93, 96, 98, 99, 100, 103, 104, 105,
106, 107, 109, 110, 111, 112, 114, 146, 160, 161, 162, 163, 164,
165, 166, 167, 169, 170, 172, 174, 175, 179, 180, 181, 182, 183,
184, 186, 187, 188, 189, 191, 192, 193, 194, 197, 202, 211, 212,
214, 215, 216, 218, 219, 220, 221, 222, 224, 227, 228, 238, 239,
241, 242, 246, 247, 248, 249, 250, 258, 259, 261, 263, 265, 266,
267, 268, 271, 274, 275, 280, 281, 282, 283, 284, 285, 286, 290,
293, 294, 298, 299, 300, 304, 308, 309, 313, 314, 315, 316, 317,
318, 319, 321, 322, 324, 325 PI3K .beta. 8, 9, 10, 11, 14, 21, 3,
12, 13, 23, 25, 53, 7, 62, 66, 82, 89, 90, 101, 142, 155, 156, 22,
24, 26, 27, 28, 29, 55, 58, 61, 63, 65, 67, 95, 97, 100, 102, 150,
157, 200, 253, 254, 34, 35, 36, 37, 38, 39, 71, 72, 74, 75, 77, 81,
153, 159, 176, 185, 255, 256, 257, 260, 40, 41, 42, 43, 44, 46, 82,
83, 84, 85, 86, 96, 201, 204, 208, 213, 262, 264, 268, 270, 52, 54,
56, 57, 59, 60, 99, 106, 108, 110, 227, 237, 251, 252, 272, 273,
278, 279, 64, 68, 69, 70, 73, 76, 111, 113, 114, 115, 267, 276,
277, 290, 287, 288, 289, 291, 78, 79, 80, 87, 88, 91, 145, 147,
149, 151, 292, 293 320, 323, 93, 98, 103, 104, 105, 154, 158, 160,
161, 107, 109, 112, 146, 167, 168, 171, 173, 152, 162, 163, 164,
174, 177, 178, 190, 165, 166, 169, 170, 191, 198, 202, 203, 172,
175, 179, 180, 205, 206, 207, 209, 181, 182, 183, 184, 210, 211,
212, 214, 186, 187, 188, 189, 215, 219, 220, 223, 192, 193, 194,
197, 228, 235, 240, 243, 216, 217, 218, 221, 244, 247, 249, 265,
222, 224, 238, 248, 269, 274, 281, 295, 259, 261, 263, 266, 296,
298, 300, 308, 271, 275, 280, 282, 316, 324 283, 284, 285, 286,
294, 299, 304, 310, 311, 312, 315, 317, 321, 322, 325 B cell
proliferation 38, 162, 199 1, 2, 5, 22, 26, 27, 39, 4, 8, 9, 10,
11, 14, 15, 3, 6, 7, 12, 13, 16, 17, EC.sub.50 (nM) 40, 43, 49, 57,
71, 87, 18, 19, 20, 21, 24, 25, 23, 33, 37, 44, 48, 53, 112, 197,
207, 235 28, 29, 30, 31, 32, 34, 54, 55, 62, 63, 66, 67, 35, 36,
41, 42, 45, 46, 68, 72, 73, 74, 75, 81, 47, 50, 51, 61, 69, 70, 82,
83, 84, 88, 89, 90, 76, 77, 78, 79, 80, 85, 93, 95, 96, 97, 99, 86,
91, 98, 100, 103, 101, 102, 108, 109, 104, 105, 106, 107, 113, 115,
123, 125, 110, 111, 114, 119, 126, 128, 134, 136, 124, 133, 135,
145, 137, 138, 139, 141, 152, 161, 162, 163, 142, 144, 146, 147,
169, 195, 212, 243, 148, 149, 150, 151, 294, 312 153, 154, 155,
156, 157, 158, 159, 160, 166, 167, 168, 170, 171, 173, 174, 176,
177, 178, 180, 187, 185, 188, 190, 191. 196, 198, 200, 201, 202,
203, 204, 205, 206, 208, 209, 210, 211, 213, 214, 215, 216, 219,
220, 221, 222, 223, 224, 227, 228, 229, 230, 231, 232, 233, 234,
237, 244, 245, 247, 248, 249, 251, 252, 253, 254, 255, 256, 257,
270, 276, 277, 278, 289, 290, 292, 295, 296, 298, 300, 301, 302,
303, 306, 308, 310, 311
TABLE-US-00004 TABLE 4 Structures of the Compounds for the IC50
results described in Table 3. Structure ##STR00341## Compound 1
##STR00342## Compound 2 ##STR00343## Compound 3 ##STR00344##
Compound 4 ##STR00345## Compound 5 ##STR00346## Compound 6
##STR00347## Compound 7 ##STR00348## Compound 8 ##STR00349##
Compound 9 ##STR00350## Compound 10 ##STR00351## Compound 11
##STR00352## Compound 12 ##STR00353## Compound 13 ##STR00354##
Compound 14 ##STR00355## Compound 15 ##STR00356## Compound 16
##STR00357## Compound 17 ##STR00358## Compound 18 ##STR00359##
Compound 19 ##STR00360## Compound 20 ##STR00361## Compound 21
##STR00362## Compound 22 ##STR00363## Compound 23 ##STR00364##
Compound 24 ##STR00365## Compound 25 ##STR00366## Compound 26
##STR00367## Compound 27 ##STR00368## Compound 28 ##STR00369##
Compound 29 ##STR00370## Compound 30 ##STR00371## Compound 31
##STR00372## Compound 32 ##STR00373## Compound 33 ##STR00374##
Compound 34 ##STR00375## Compound 35 ##STR00376## Compound 36
##STR00377## Compound 37 ##STR00378## Compound 38 ##STR00379##
Compound 39 ##STR00380## Compound 40 ##STR00381## Compound 41
##STR00382## Compound 42 ##STR00383## Compound 43 ##STR00384##
Compound 44 ##STR00385## Compound 45 ##STR00386## Compound 46
##STR00387## Compound 47 ##STR00388## Compound 48 ##STR00389##
Compound 49 ##STR00390## Compound 50 ##STR00391## Compound 51
##STR00392## Compound 52 ##STR00393## Compound 53 ##STR00394##
Compound 54 ##STR00395## Compound 55 ##STR00396## Compound 56
##STR00397## Compound 57 ##STR00398## Compound 58 ##STR00399##
Compound 59 ##STR00400## Compound 60 ##STR00401## Compound 61
##STR00402## Compound 62 ##STR00403## Compound 63 ##STR00404##
Compound 64 ##STR00405## Compound 65 ##STR00406## Compound 66
##STR00407## Compound 67 ##STR00408## Compound 68 ##STR00409##
Compound 69 ##STR00410## Compound 70 ##STR00411## Compound 71
##STR00412## Compound 72 ##STR00413## Compound 73 ##STR00414##
Compound 74 ##STR00415## Compound 75 ##STR00416## Compound 76
##STR00417## Compound 77 ##STR00418## Compound 78 ##STR00419##
Compound 79 ##STR00420## Compound 80 ##STR00421## Compound 81
##STR00422## Compound 82 ##STR00423## Compound 83 ##STR00424##
Compound 84 ##STR00425## Compound 85 ##STR00426## Compound 86
##STR00427## Comopund 87 ##STR00428## Compound 88 ##STR00429##
Compound 89 ##STR00430## Compound 90 ##STR00431## Compound 91
##STR00432## Compound 92 ##STR00433## Compound 93 ##STR00434##
Compound 94 ##STR00435## Compound 95 ##STR00436## Compound 96
##STR00437## Compound 97 ##STR00438## Compound 98 ##STR00439##
Compound 99 ##STR00440## Compound 100 ##STR00441## Compound 101
##STR00442## Compound 102 ##STR00443## Compound 103 ##STR00444##
Compound 104 ##STR00445## Compound 105 ##STR00446## Compound 106
##STR00447## Compound 107 ##STR00448## Compound 108 ##STR00449##
Compound 109 ##STR00450## Compound 110 ##STR00451## Compound 111
##STR00452## Compound 112 ##STR00453## Compound 113 ##STR00454##
Compound 114 ##STR00455## Compound 115 ##STR00456## Compound 116
##STR00457## Compound 117 ##STR00458## Compound 118 ##STR00459##
Compound 119 ##STR00460## Compound 120 ##STR00461## Compound 121
##STR00462## Compound 122 ##STR00463## Compound 123
##STR00464## Compound 124 ##STR00465## Compound 125 ##STR00466##
Compound 126 ##STR00467## Compound 127 ##STR00468## Compound 128
##STR00469## Compound 129 ##STR00470## Compound 130 ##STR00471##
Compound 131 ##STR00472## Compound 132 ##STR00473## Compound 133
##STR00474## Compound 134 ##STR00475## Compound 135 ##STR00476##
Compound 136 ##STR00477## Compound 137 ##STR00478## Compound 138
##STR00479## Compound 139 ##STR00480## Compound 141 ##STR00481##
Compound 142 ##STR00482## Compound 143 ##STR00483## Compound 144
##STR00484## Compound 145 ##STR00485## Compound 146 ##STR00486##
Compound 147 ##STR00487## Compound 148 ##STR00488## Compound 149
##STR00489## Compound 150 ##STR00490## Compound 151 ##STR00491##
Compound 152 ##STR00492## Compound 153 ##STR00493## Compound 154
##STR00494## Compound 155 ##STR00495## Compound 156 ##STR00496##
Compound 157 ##STR00497## Compound 158 ##STR00498## Compound 159
##STR00499## Compound 160 ##STR00500## Compound 161 ##STR00501##
Compound 162 ##STR00502## Compound 163 ##STR00503## Compound 164
##STR00504## Compound 165 ##STR00505## Compound 166 ##STR00506##
Compound 167 ##STR00507## Compound 168 ##STR00508## Compound 169
##STR00509## Compound 170 ##STR00510## Compound 171 ##STR00511##
Compound 172 ##STR00512## Compound 173 ##STR00513## Compound 174
##STR00514## Compound 175 ##STR00515## Compound 176 ##STR00516##
Compound 177 ##STR00517## Compound 178 ##STR00518## Compound 179
##STR00519## Compound 180 ##STR00520## Compound 181 ##STR00521##
Compound 182 ##STR00522## Compound 183 ##STR00523## Compound 184
##STR00524## Compound 185 ##STR00525## Compound 186 ##STR00526##
Compound 187 ##STR00527## Compound 188 ##STR00528## Compound 189
##STR00529## Compound 190 ##STR00530## Compound 191 ##STR00531##
Compound 192 ##STR00532## Compound 193 ##STR00533## Compound 194
##STR00534## Compound 195 ##STR00535## Compound 196 ##STR00536##
Compound 197 ##STR00537## Compound 198 ##STR00538## Compound 199
##STR00539## Compound 200 ##STR00540## Compound 201 ##STR00541##
Compound 202 ##STR00542## Compound 203 ##STR00543## Compound 204
##STR00544## Compound 205 ##STR00545## Compound 206 ##STR00546##
Compound 207 ##STR00547## Compound 208 ##STR00548## Compound 209
##STR00549## Compound 210 ##STR00550## Compound 211 ##STR00551##
Compound 212 ##STR00552## Compound 213 ##STR00553## Compound 214
##STR00554## Compound 215 ##STR00555## Compound 216 ##STR00556##
Compound 217 ##STR00557## Compound 218 ##STR00558## Compound 219
##STR00559## Compound 220 ##STR00560## Compound 221 ##STR00561##
Compound 222 ##STR00562## Compound 223 ##STR00563## Compound 224
##STR00564## Compound 225 ##STR00565## Compound 226 ##STR00566##
Compound 227 ##STR00567## Compound 228 ##STR00568## Compound 229
##STR00569## Compound 230 ##STR00570## Compound 231 ##STR00571##
Compound 232 ##STR00572## Compound 233 ##STR00573## Compound 234
##STR00574## Compound 235 ##STR00575## Compound 236 ##STR00576##
Compound 237 ##STR00577## Compound 238 ##STR00578## Compound 239
##STR00579## Compound 240 ##STR00580## Compound 241 ##STR00581##
Compound 242 ##STR00582## Compound 243 ##STR00583## Compound 244
##STR00584## Compound 245 ##STR00585## Compound 246 ##STR00586##
Compound 247 ##STR00587## Compound 248 ##STR00588## Compound
249
##STR00589## Compound 250 ##STR00590## Compound 251 ##STR00591##
Compound 252 ##STR00592## Compound 253 ##STR00593## Compound 254
##STR00594## Compound 255 ##STR00595## Compound 256 ##STR00596##
Compound 257 ##STR00597## Compound 258 ##STR00598## Compound 259
##STR00599## Compound 260 ##STR00600## Compound 261 ##STR00601##
Compound 262 ##STR00602## Compound 263 ##STR00603## Compound 264
##STR00604## Compound 265 ##STR00605## Compound 266 ##STR00606##
Compound 267 ##STR00607## Compound 268 ##STR00608## Compound 269
##STR00609## Compound 270 ##STR00610## Compound 271 ##STR00611##
Compound 272 ##STR00612## Compound 273 ##STR00613## Compound 274
##STR00614## Compound 275 ##STR00615## Compound 276 ##STR00616##
Compound 277 ##STR00617## Compound 278 ##STR00618## Compound 279
##STR00619## Compound 280 ##STR00620## Compound 281 ##STR00621##
Compound 282 ##STR00622## Compound 283 ##STR00623## Compound 284
##STR00624## Compound 285 ##STR00625## Compound 286 ##STR00626##
Compound 287 ##STR00627## Compound 288 ##STR00628## Compound 289
##STR00629## Compound 290 ##STR00630## Compound 291 ##STR00631##
Compound 292 ##STR00632## Compound 293 ##STR00633## Compound 294
##STR00634## Compound 295 ##STR00635## Compound 296 ##STR00636##
Compound 297 ##STR00637## Compound 298 ##STR00638## Compound 299
##STR00639## Compound 300 ##STR00640## Compound 301 ##STR00641##
Compound 302 ##STR00642## Compound 303 ##STR00643## Compound 304
##STR00644## Compound 305 ##STR00645## Compound 306 ##STR00646##
Compound 307 ##STR00647## Compound 308 ##STR00648## Compound 309
##STR00649## Compound 310 ##STR00650## Compound 311 ##STR00651##
Compound 312 ##STR00652## Compound 313 ##STR00653## Compound 314
##STR00654## Compound 315 ##STR00655## Compound 316 ##STR00656##
Compound 317 ##STR00657## Compound 318 ##STR00658## Compound 319
##STR00659## Compound 320 ##STR00660## Compound 321 ##STR00661##
Compound 322 ##STR00662## Compound 323 ##STR00663## Compound 324
##STR00664## Compound 325
Example 22: Expression and Inhibition Assays of
p110.alpha./p85.alpha., p110.beta./p85.alpha.,
p110.delta./p85.alpha., and p110.gamma.:
Class I PI3-Ks can be either purchased (p110.alpha./p85.alpha.,
p110.beta./p85.alpha., p110.delta./p85.alpha. from Upstate, and
p110.gamma. from Sigma) or expressed as previously described
(Knight et al., 2004). IC50 values are measured using either a
standard TLC assay for lipid kinase activity (described below) or a
high-throughput membrane capture assay. Kinase reactions are
performed by preparing a reaction mixture containing kinase,
inhibitor (2% DMSO final concentration), buffer (25 mM HEPES, pH
7.4, 10 mM MgCl2), and freshly sonicated phosphatidylinositol (100
.mu.g/ml). Reactions are initiated by the addition of ATP
containing 10 .mu.Ci of .gamma.-32P-ATP to a final concentration 10
or 100 .mu.M and allowed to proceed for 5 minutes at room
temperature. For TLC analysis, reactions are then terminated by the
addition of 105 .mu.l 1N HCl followed by 160 .mu.l CHCl3:MeOH
(1:1). The biphasic mixture is vortexed, briefly centrifuged, and
the organic phase is transferred to a new tube using a gel loading
pipette tip precoated with CHCl.sub.3. This extract is spotted on
TLC plates and developed for 3-4 hours in a 65:35 solution of
n-propanol:1M acetic acid. The TLC plates are then dried, exposed
to a phosphorimager screen (Storm, Amersham), and quantitated. For
each compound, kinase activity is measured at 10-12 inhibitor
concentrations representing two-fold dilutions from the highest
concentration tested (typically, 200 .mu.M). For compounds showing
significant activity, IC50 determinations are repeated two to four
times, and the reported value is the average of these independent
measurements.
Other commercial kits or systems for assaying PI3-K activities are
available. The commercially available kits or systems can be used
to screen for inhibitors and/or agonists of PI3-Ks including but
not limited to PI 3-Kinase .alpha., .beta., .delta., and .gamma..
An exemplary system is PI 3-Kinase (human) HTRF.TM. Assay from
Upstate. The assay can be carried out according to the procedures
suggested by the manufacturer. Briefly, the assay is a time
resolved FRET assay that indirectly measures PIP3 product formed by
the activity of a PI3-K. The kinase reaction is performed in a
microtitre plate (e.g., a 384 well microtitre plate). The total
reaction volume is approximately 20 ul per well. In the first step,
each well receives 2 ul of test compound in 20% dimethylsulphoxide
resulting in a 2% DMSO final concentration. Next, approximately
14.5 ul of a kinase/PIP2 mixture (diluted in 1.times. reaction
buffer) is added per well for a final concentration of 0.25-0.3
ug/ml kinase and 10 uM PIP2. The plate is sealed and incubated for
15 minutes at room temperature. To start the reaction, 3.5 ul of
ATP (diluted in 1.times. reaction buffer) is added per well for a
final concentration of 10 uM ATP. The plate is sealed and incubated
for 1 hour at room temperature. The reaction is stopped by adding 5
ul of Stop Solution per well and then 5 ul of Detection Mix is
added per well. The plate is sealed, incubated for 1 hour at room
temperature, and then read on an appropriate plate reader. Data is
analyzed and IC50s are generated using GraphPad Prism 5.
Example 23: Expression and Inhibition Assays of Abl
The cross-activity or lack thereof of one or more compounds of the
present invention against Abl kinase can be measured according to
any procedures known in the art or methods disclosed below. For
example, the compounds described herein can be assayed in
triplicate against recombinant full-length Abl or Abl (T315I)
(Upstate) in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgCl2,
200 .mu.M ATP (2.5 .mu.Ci of .gamma.-32P-ATP), and 0.5 mg/mL BSA.
The optimized Abl peptide substrate EAIYAAPFAKKK (SEQ ID NO: 1) is
used as phosphoacceptor (200 .mu.M). Reactions are terminated by
spotting onto phosphocellulose sheets, which are washed with 0.5%
phosphoric acid (approximately 6 times, 5-10 minutes each). Sheets
are dried and the transferred radioactivity quantitated by
phosphorimaging.
Example 24: Expression and Inhibition Assays of Hck
The cross-activity or lack thereof of one or more compounds of the
present invention against Hck kinase can be measured according to
any procedures known in the art or methods disclosed below. The
compounds described herein can be assayed in triplicate against
recombinant full-length Hck in an assay containing 25 mM HEPES, pH
7.4, 10 mM MgCl2, 200 .mu.M ATP (2.5 .mu.Ci of .gamma.-32P-ATP),
and 0.5 mg/mL BSA. The optimized Src family kinase peptide
substrate EIYGEFKKK (SEQ ID NO: 2) is used as phosphoacceptor (200
.mu.M). Reactions are terminated by spotting onto phosphocellulose
sheets, which are washed with 0.5% phosphoric acid (approximately 6
times, 5-10 minutes each). Sheets are dried and the transferred
radioactivity quantitated by phosphorimaging.
Example 25: Expression and Inhibition Assays of Inulsin Receptor
(IR)
The cross-activity or lack thereof of one or more compounds of the
present invention against IR receptor kinase can be measured
according to any procedures known in the art or methods disclosed
below. The compounds described herein can be assayed in triplicate
against recombinant insulin receptor kinase domain (Upstate) in an
assay containing 25 mM HEPES, pH 7.4, 10 mM MgCl2, 10 mM MnCl2, 200
.mu.M ATP (2.5 .mu.Ci of .gamma.-32P-ATP), and 0.5 mg/mL BSA. Poly
E-Y (Sigma; 2 mg/mL) is used as a substrate. Reactions are
terminated by spotting onto nitrocellulose, which is washed with 1M
NaCl/1% phosphoric acid (approximately 6 times, 5-10 minutes each).
Sheets are dried and the transferred radioactivity quantitated by
phosphorimaging.
Example 26: Expression and Inhibition Assays of Src
The cross-activity or lack thereof of one or more compounds of the
present invention against Src kinase can be measured according to
any procedures known in the art or methods disclosed below. The
compounds described herein can be assayed in triplicate against
recombinant full-length Src or Src (T338I) in an assay containing
25 mM HEPES, pH 7.4, 10 mM MgCl2, 200 .mu.M ATP (2.5 .mu.Ci of
.gamma.-32P-ATP), and 0.5 mg/mL BSA. The optimized Src family
kinase peptide substrate EIYGEFKKK (SEQ ID NO: 2) is used as
phosphoacceptor (200 .mu.M). Reactions are terminated by spotting
onto phosphocellulose sheets, which are washed with 0.5% phosphoric
acid (approximately 6 times, 5-10 minutes each). Sheets were dried
and the transferred radioactivity quantitated by
phosphorimaging.
Example 27: Expression and Inhibition Assays of DNA-PK (DNAK)
The cross-activity or lack thereof of one or more compounds of the
present invention against DNAK kinase can be measured according to
any procedures known in the art. DNA-PK can be purchased from
Promega and assayed using the DNA-PK Assay System (Promega)
according to the manufacturer's instructions.
Example 28: Expression and Inhibition Assays of mTOR
The cross-activity or lack thereof of one or more compounds of the
present invention against mTor can be measured according to any
procedures known in the art or methods disclosed below. The
compounds described herein can be tested against recombinant mTOR
(Invitrogen) in an assay containing 50 mM HEPES, pH 7.5, 1 mM EGTA,
10 mM MgCl2, 2.5 mM, 0.01% Tween, 10 .mu.M ATP (2.5 .mu.Ci of
.mu.-32P-ATP), and 3 .mu.g/mL BSA. Rat recombinant PHAS-1/4EBP1
(Calbiochem; 2 mg/mL) is used as a substrate. Reactions are
terminated by spotting onto nitrocellulose, which is washed with 1M
NaCl/1% phosphoric acid (approximately 6 times, 5-10 minutes each).
Sheets are dried and the transferred radioactivity quantitated by
phosphorimaging.
Other kits or systems for assaying mTOR activity are commercially
available. For instance, one can use Invitrogen's LanthaScreen.TM.
Kinase assay to test the inhibitors of mTOR disclosed herein. This
assay is a time resolved FRET platform that measures the
phosphorylation of GFP labeled 4EBP1 by mTOR kinase. The kinase
reaction is performed in a white 384 well microtitre plate. The
total reaction volume is 20 ul per well and the reaction buffer
composition is 50 mM HEPES pH7.5, 0.01% Polysorbate 20, 1 mM EGTA,
10 mM MnCl2, and 2 mM DTT. In the first step, each well receives 2
ul of test compound in 20% dimethylsulphoxide resulting in a 2%
DMSO final concentration. Next, 8 ul of mTOR diluted in reaction
buffer is added per well for a 60 ng/ml final concentration. To
start the reaction, 10 ul of an ATP/GFP-4EBP1 mixture (diluted in
reaction buffer) is added per well for a final concentration of 10
uM ATP and 0.5 uM GFP-4EBP1. The plate is sealed and incubated for
1 hour at room temperature. The reaction is stopped by adding 10 ul
per well of a Tb-anti-pT46 4EBP1 antibody/EDTA mixture (diluted in
TR-FRET buffer) for a final concentration of 1.3 nM antibody and
6.7 mM EDTA. The plate is sealed, incubated for 1 hour at room
temperature, and then read on a plate reader set up for
LanthaScreen.TM. TR-FRET. Data is analyzed and IC50s are generated
using GraphPad Prism 5.
Example 29: Expression and Inhibition Assays of Vascular
Endothelial Growth Receptor
The cross-activity or lack thereof of one or more compounds of the
present invention against VEGF receptor can be measured according
to any procedures known in the art or methods disclosed below. The
compounds described herein can be tested against recombinant KDR
receptor kinase domain (Invitrogen) in an assay containing 25 mM
HEPES, pH 7.4, 10 mM MgCl2, 0.1% BME, 10 .mu.M ATP (2.5 .mu.Ci of
.mu.-32P-ATP), and 3 .mu.g/mL BSA. Poly E-Y (Sigma; 2 mg/mL) is
used as a substrate. Reactions are terminated by spotting onto
nitrocellulose, which is washed with 1M NaCl/1% phosphoric acid
(approximately 6 times, 5-10 minutes each). Sheets are dried and
the transferred radioactivity quantitated by phosphorimaging.
Example 30: Expression and Inhibition Assays of Ephrin Receptor B4
(EphB4)
The cross-activity or lack thereof of one or more compounds of the
present invention against EphB4 can be measured according to any
procedures known in the art or methods disclosed below. The
compounds described herein can be tested against recombinant Ephrin
receptor B4 kinase domain (Invitrogen) in an assay containing 25 mM
HEPES, pH 7.4, 10 mM MgCl2, 0.1% BME, 10 .mu.M ATP (2.5 .mu.Ci of
.mu.-32P-ATP), and 3 .mu.g/mL BSA. Poly E-Y (Sigma; 2 mg/mL) is
used as a substrate. Reactions are terminated by spotting onto
nitrocellulose, which is washed with 1M NaCl/1% phosphoric acid
(approximately 6 times, 5-10 minutes each). Sheets are dried and
the transferred radioactivity quantitated by phosphorimaging.
Example 31: Expression and Inhibition Assays of Epidermal Growth
Factor Receptor (EGFR)
The cross-activity or lack thereof of one or more compounds of the
present invention against EGFR kinase can be measured according to
any procedures known in the art or methods disclosed below. The
compounds described herein can be tested against recombinant EGF
receptor kinase domain (Invitrogen) in an assay containing 25 mM
HEPES, pH 7.4, 10 mM MgCl2, 0.1% BME, 10 .mu.M ATP (2.5 .mu.Ci of
.mu.-32P-ATP), and 3 .mu.g/mL BSA. Poly E-Y (Sigma; 2 mg/mL) is
used as a substrate. Reactions are terminated by spotting onto
nitrocellulose, which is washed with 1M NaCl/1% phosphoric acid
(approximately 6 times, 5-10 minutes each). Sheets are dried and
the transferred radioactivity quantitated by phosphorimaging.
Example 32: Expression and Inhibition Assays of KIT Assay
The cross-activity or lack thereof of one or more compounds of the
present invention against KIT kinase can be measured according to
any procedures known in the art or methods disclosed below. The
compounds described herein can be tested against recombinant KIT
kinase domain (Invitrogen) in an assay containing 25 mM HEPES, pH
7.4, 10 mM MgCl2, 1 mM DTT, 10 mM MnCl2, 10 .mu.M ATP (2.5 .mu.Ci
of .mu.-32P-ATP), and 3 .mu.g/mL BSA. Poly E-Y (Sigma; 2 mg/mL) is
used as a substrate. Reactions are terminated by spotting onto
nitrocellulose, which is washed with 1M NaCl/1% phosphoric acid
(approximately 6 times, 5-10 minutes each). Sheets are dried and
the transferred radioactivity quantitated by phosphorimaging.
Example 33: Expression and Inhibition Assays of RET
The cross-activity or lack thereof of one or more compounds of the
present invention against RET kinase can be measured according to
any procedures known in the art or methods disclosed below. The
compounds described herein can be tested against recombinant RET
kinase domain (Invitrogen) in an assay containing 25 mM HEPES, pH
7.4, 10 mM MgCl2, 2.5 mM DTT,10 .mu.M ATP (2.5 .mu.Ci of
.mu.-32P-ATP), and 3 .mu.g/mL BSA. The optimized Abl peptide
substrate EAIYAAPFAKKK (SEQ ID NO: 1) is used as phosphoacceptor
(200 .mu.M). Reactions are terminated by spotting onto
phosphocellulose sheets, which are washed with 0.5% phosphoric acid
(approximately 6 times, 5-10 minutes each). Sheets are dried and
the transferred radioactivity quantitated by phosphorimaging.
Example 34: Expression and Inhibition Assays of Platelet Derived
Growth Factor Receptor (PDGFR)
The cross-activity or lack thereof of one or more compounds of the
present invention against PDGFR kinase can be measured according to
any procedures known in the art or methods disclosed below. The
compounds described herein can be tested against recombinant PDG
receptor kinase domain (Invitrogen) in an assay containing 25 mM
HEPES, pH 7.4, 10 mM MgCl2, 2.5 mM DTT,10 .mu.M ATP (2.5 .mu.Ci of
.mu.-32P-ATP), and 3 .mu.g/mL BSA. The optimized Abl peptide
substrate EAIYAAPFAKKK (SEQ ID NO: 1) is used as phosphoacceptor
(200 .mu.M). Reactions are terminated by spotting onto
phosphocellulose sheets, which are washed with 0.5% phosphoric acid
(approximately 6 times, 5-10 minutes each). Sheets are dried and
the transferred radioactivity quantitated by phosphorimaging.
Example 35: Expression and Inhibition Assays of FMS-Related
Tyrosine Kinase 3 (FLT-3)
The cross-activity or lack thereof of one or more compounds of the
present invention against FLT-3 kinase can be measured according to
any procedures known in the art or methods disclosed below. The
compounds described herein can be tested against recombinant FLT-3
kinase domain (Invitrogen) in an assay containing 25 mM HEPES, pH
7.4, 10 mM MgCl2, 2.5 mM DTT,10 .mu.M ATP (2.5 .mu.Ci of
.mu.-32P-ATP), and 3 .mu.g/mL BSA. The optimized Abl peptide
substrate EAIYAAPFAKKK (SEQ ID NO: 1) is used as phosphoacceptor
(200 .mu.M). Reactions are terminated by spotting onto
phosphocellulose sheets, which are washed with 0.5% phosphoric acid
(approximately 6 times, 5-10 minutes each). Sheets are dried and
the transferred radioactivity quantitated by phosphorimaging.
Example 36: Expression and Inhibition Assays of TEK Receptor
Tyrosine Kinase (TIE2)
The cross-activity or lack thereof of one or more compounds of the
present invention against TIE2 kinase can be measured according to
any procedures known in the art or methods disclosed below. The
compounds described herein can be tested against recombinant TIE2
kinase domain (Invitrogen) in an assay containing 25 mM HEPES, pH
7.4, 10 mM MgCl2, 2 mM DTT, 10 mM MnCl2, 10 .mu.M ATP (2.5 .mu.Ci
of .mu.-32P-ATP), and 3 .mu.g/mL BSA. Poly E-Y (Sigma; 2 mg/mL) is
used as a substrate. Reactions are terminated by spotting onto
nitrocellulose, which is washed with 1M NaCl/1% phosphoric acid
(approximately 6 times, 5-10 minutes each). Sheets are dried and
the transferred radioactivity quantitated by phosphorimaging.
Example 37: B Cell Activation and Proliferation Assay
The ability of one or more subject compounds to inhibit B cell
activation and proliferation is determined according to standard
procedures known in the art. For example, an in vitro cellular
proliferation assay is established that measures the metabolic
activity of live cells. The assay is performed in a 96 well
microtiter plate using Alamar Blue reduction. Balb/c splenic B
cells are purified over a Ficoll-Paque.TM. PLUS gradient followed
by magnetic cell separation using a MACS B cell Isolation Kit
(Miletenyi). Cells are plated in 90 ul at 50,000 cells/well in B
Cell Media (RPMI+10% FBS+Penn/Strep+50 uM bME+5 mM HEPES). A
compound disclosed herein is diluted in B Cell Media and added in a
10 ul volume. Plates are incubated for 30 min at 37 C and 5%
CO.sub.2 (0.2% DMSO final concentration). A 50 ul B cell
stimulation cocktail is then added containing either 10 ug/ml LPS
or 5 ug/ml F(ab')2 Donkey anti-mouse IgM plus 2 ng/ml recombinant
mouse IL4 in B Cell Media. Plates are incubated for 72 hours at
37.degree. C. and 5% CO.sub.2. A volume of 15 uL of Alamar Blue
reagent is added to each well and plates are incubated for 5 hours
at 37 C and 5% CO.sub.2. Alamar Blue fluoresce is read at
560Ex/590Em, and IC50 or EC50 values are calculated using GraphPad
Prism 5.
Example 38: Tumor Cell Line Proliferation Assay
The ability of one or more subject compounds to inhibit tumor cell
line proliferation is determined according to standard procedures
known in the art. For instance, an in vitro cellular proliferation
assay can be performed to measure the metabolic activity of live
cells. The assay is performed in a 96 well microtiter plate using
Alamar Blue reduction. Human tumor cell lines are obtained from
ATCC (e.g., MCF7, U-87 MG, MDA-MB-468, PC-3), grown to confluency
in T75 flasks, trypsinized with 0.25% trypsin, washed one time with
Tumor Cell Media (DMEM+10% FBS), and plated in 90 ul at 5,000
cells/well in Tumor Cell Media. A compound disclosed herein is
diluted in Tumor Cell Media and added in a 10 ul volume. Plates are
incubated for 72 hours at 37 C and 5% CO.sub.2. A volume of 10 uL
of Alamar Blue reagent is added to each well and plates are
incubated for 3 hours at 37 C and 5% CO.sub.2. Alamar Blue
fluoresce is read at 560Ex/590Em, and IC50 values are calculated
using GraphPad Prism 5.
Example 39: Antitumor Activity In Vivo
The compounds described herein can be evaluated in a panel of human
and murine tumor models.
Paclitaxel-Refractory Tumor Models
1. Clinically-Derived Ovarian Carcinoma Model.
This tumor model is established from a tumor biopsy of an ovarian
cancer patient. Tumor biopsy is taken from the patient.
The compounds described herein are administered to nude mice
bearing staged tumors using an every 2 days.times.5 schedule.
2. A2780Tax Human Ovarian Carcinoma Xenograft (Mutated
Tubulin).
A2780Tax is a paclitaxel-resistant human ovarian carcinoma model.
It is derived from the sensitive parent A2780 line by co-incubation
of cells with paclitaxel and verapamil, an MDR-reversal agent. Its
resistance mechanism has been shown to be non-MDR related and is
attributed to a mutation in the gene encoding the beta-tubulin
protein.
The compounds described herein can be administered to mice bearing
staged tumors on an every 2 days.times.5 schedule.
3. HCT116/VM46 Human Colon Carcinoma Xenograft (Multi-Drug
Resistant).
HCT116/VM46 is an MDR-resistant colon carcinoma developed from the
sensitive HCT116 parent line. In vivo, grown in nude mice,
HCT116/VM46 has consistently demonstrated high resistance to
paclitaxel.
The compounds described herein can be administered to mice bearing
staged tumors on an every 2 days.times.5 schedule.
5. M5076 Murine Sarcoma Model
M5076 is a mouse fibrosarcoma that is inherently refractory to
paclitaxel in vivo.
The compounds described herein can be administered to mice bearing
staged tumors on an every 2 days.times.5 schedule.
One or more compounds of the invention can be used in combination
other therapeutic agents in vivo in the multidrug resistant human
colon carcinoma xenografts HCT/VM46 or any other model known in the
art including those described herein.
Example 40: Microsome Stability Assay
The stability of one or more subject compounds is determined
according to standard procedures known in the art. For example,
stability of one or more subject compounds is established by an in
vitro assay. In particular, an in vitro microsome stability assay
is established that measures stability of one or more subject
compounds when reacting with mouse, rat or human microsomes from
liver. The microsome reaction with compounds is performed in 1.5 mL
Eppendorf tube. Each tube contains 0.1 .mu.L of 10.0 mg/ml NADPH;
75 .mu.L of 20.0 mg/ml mouse, rat or human liver microsome; 0.4
.mu.L of 0.2 M phosphate buffer, and 425 .mu.L of ddH.sub.2O.
Negative control (without NADPH) tube contains 75 .mu.L of 20.0
mg/ml mouse, rat or human liver microsome; 0.4 .mu.L of 0.2 M
phosphate buffer, and 525 .mu.L of ddH.sub.2O. The reaction is
started by adding 1.0 .mu.L of 10.0 mM tested compound. The
reaction tubes are incubated at 37.degree. C. 100 .mu.L sample is
collected into new Eppendorf tube containing 300 .mu.L cold
Methanol at 0, 5, 10, 15, 30 and 60 minutes of reaction. Samples
are centrifuged at 15,000 rpm to remove protein. Supernatant of
centrifuged sample is transferred to new tube. Concentration of
stable compound after reaction with microsome in the supernatant is
measured by Liquid Chromatography/Mass Spectrometry (LC-MS).
Example 41: Plasma Stability Assay
The stability of one or more subject compounds in plasma is
determined according to standard procedures known in the art. See,
e.g., Rapid Commun. Mass Spectrom., 10: 1019-1026. The following
procedure is an HPLC-MS/MS assay using human plasma; other species
including monkey, dog, rat, and mouse are also available. Frozen,
heparinized human plasma is thawed in a cold water bath and spun
for 10 minutes at 2000 rpm at 4.degree. C. prior to use. A subject
compound is added from a 400 .mu.M stock solution to an aliquot of
pre-warmed plasma to give a final assay volume of 400 .mu.L (or 800
.mu.L for half-life determination), containing 5 .mu.M test
compound and 0.5% DMSO. Reactions are incubated, with shaking, for
0 minutes and 60 minutes at 37.degree. C., or for 0, 15, 30, 45 and
60 minutes at 37 C for half life determination. Reactions are
stopped by transferring 50 .mu.L of the incubation mixture to 200
.mu.L of ice-cold acetonitrile and mixed by shaking for 5 minutes.
The samples are centrifuged at 6000.times.g for 15 minutes at
4.degree. C. and 120 .mu.L of supernatant removed into clean tubes.
The samples are then evaporated to dryness and submitted for
analysis by HPLC-MS/MS.
Where desired, one or more control or reference compounds (5 .mu.M)
are tested simultaneously with the test compounds: one compound,
propoxycaine, with low plasma stability and another compound,
propantheline, with intermediate plasma stability.
Samples are reconstituted in acetonitrile/methanol/water (1/1/2,
v/v/v) and analyzed via (RP)HPLC-MS/MS using selected reaction
monitoring (SRM). The HPLC conditions consist of a binary LC pump
with autosampler, a mixed-mode, C12, 2.times.20 mm column, and a
gradient program. Peak areas corresponding to the analytes are
recorded by HPLC-MS/MS. The ratio of the parent compound remaining
after 60 minutes relative to the amount remaining at time zero,
expressed as percent, is reported as plasma stability. In case of
half-life determination, the half-life is estimated from the slope
of the initial linear range of the logarithmic curve of compound
remaining (%) vs. time, assuming first order kinetics.
Example 42: Chemical Stability
The chemical stability of one or more subject compounds is
determined according to standard procedures known in the art. The
following details an exemplary procedure for ascertaining chemical
stability of a subject compound. The default buffer used for the
chemical stability assay is phosphate-buffered saline (PBS) at pH
7.4; other suitable buffers can be used. A subject compound is
added from a 100 .mu.M stock solution to an aliquot of PBS (in
duplicate) to give a final assay volume of 400 .mu.L, containing 5
.mu.M test compound and 1% DMSO (for half-life determination a
total sample volume of 700 .mu.L is prepared). Reactions are
incubated, with shaking, for 0 minutes and 24 hours at 37.degree.
C.; for half-life determination samples are incubated for 0, 2, 4,
6, and 24 hours. Reactions are stopped by adding immediately 100
.mu.L of the incubation mixture to 100 .mu.L of acetonitrile and
vortexing for 5 minutes. The samples are then stored at -20.degree.
C. until analysis by HPLC-MS/MS. Where desired, a control compound
or a reference compound such as chlorambucil (5 .mu.M) is tested
simultaneously with a subject compound of interest, as this
compound is largely hydrolyzed over the course of 24 hours. Samples
are analyzed via (RP)HPLC-MS/MS using selected reaction monitoring
(SRM). The HPLC conditions consist of a binary LC pump with
autosampler, a mixed-mode, C12, 2.times.20 mm column, and a
gradient program. Peak areas corresponding to the analytes are
recorded by HPLC-MS/MS. The ratio of the parent compound remaining
after 24 hours relative to the amount remaining at time zero,
expressed as percent, is reported as chemical stability. In case of
half-life determination, the half-life is estimated from the slope
of the initial linear range of the logarithmic curve of compound
remaining (%) vs. time, assuming first order kinetics.
Example 43: Akt Kinase Assay
Cells comprising components of the Akt/mTOR pathway, including but
not limited to L6 myoblasts, B-ALL cells, B-cells, T-cells,
leukemia cells, bone marrow cells, p190 transduced cells,
philladelphia chromosome positive cells (Ph+), and mouse embryonic
fibroblasts, are typically grown in cell growth media such as DMEM
supplemented with fetal bovine serum and/or antibiotics, and grown
to confluency.
In order to compare the effect of one or more compounds disclosed
herein on Akt activation, said cells are serum starved overnight
and incubated with one or more compounds disclosed herein or about
0.1% DMSO for approximately 1 minute to about 1 hour prior to
stimulation with insulin (e.g. 100 nM) for about 1 minutes to about
1 hour. Cells are lysed by scraping into ice cold lysis buffer
containing detergents such as sodium dodecyl sulfate and protease
inhibitors (e.g., PMSF). After contacting cells with lysis buffer,
the solution is briefly sonicated, cleared by centrifugation,
resolved by SDS-PAGE, transferred to nitrocellulose or PVDF and
immunoblotted using antibodies to phospho-Akt S473, phospho-Akt
T308, Akt, and .beta.-actin (Cell Signaling Technologies).
The results demonstrate that one or more compounds of the present
disclosure inhibit insulin stimulated phosphorylation of Akt at
S473. Alternatively, some compounds disclosed herein additionally
inhibit insulin stimulated phosphorylation of Akt at T308. Such
class of compounds can inhibit Akt more effectively than rapamycin
and may be indicative of mTORC2 inhibitors or inhibitors of
upstream kinases such as PI3K or Akt.
Example 44: Kinase Signaling in Blood
PI3K/Akt/mTor signaling is measured in blood cells using the
phosflow method (Methods Enzymol. 2007; 434:131-54). The advantage
of this method is that it is by nature a single cell assay so that
cellular heterogeneity can be detected rather than population
averages. This allows concurrent distinction of signaling states in
different populations defined by other markers. Phosflow is also
highly quantitative. To test the effects of one or more compounds
disclosed herein, unfractionated splenocytes, or peripheral blood
mononuclear cells are stimulated with anti-CD3 to initiate T-cell
receptor signaling. The cells are then fixed and stained for
surface markers and intracellular phosphoproteins. It is expected
that inhibitors disclosed herein inhibit anti-CD3 mediated
phosphorylation of Akt-S473 and S6, whereas rapamycin inhibits S6
phosphorylation and enhances Akt phosphorylation under the
conditions tested.
Similarly, aliquots of whole blood are incubated for 15 minutes
with vehicle (e.g. 0.1% DMSO) or kinase inhibitors at various
concentrations, before addition of stimuli to crosslink the T cell
receptor (TCR) (anti-CD3 with secondary antibody) or the B cell
receptor (BCR) using anti-kappa light chain antibody (Fab'2
fragments). After approximately 5 and 15 minutes, samples are fixed
(e.g. with cold 4% paraformaldehyde) and used for phosflow. Surface
staining is used to distinguish T and B cells using antibodies
directed to cell surface markers that are known to the art. The
level of phosphorylation of kinase substrates such as Akt and S6
are then measured by incubating the fixed cells with labeled
antibodies specific to the phosphorylated isoforms of these
proteins. The population of cells are then analyzed by flow
cytometry.
Example 45: Colony Formation Assay
Murine bone marrow cells freshly transformed with a p190 BCR-Abl
retrovirus (herein referred to as p190 transduced cells) are plated
in the presence of various drug combinations in M3630
methylcellulose media for about 7 days with recombinant human IL-7
in about 30% serum, and the number of colonies formed is counted by
visual examination under a microscope.
Alternatively, human peripheral blood mononuclear cells are
obtained from Philadelphia chromosome positive (Ph+) and negative
(Ph-) patients upon initial diagnosis or relapse. Live cells are
isolated and enriched for CD19+ CD34+ B cell progenitors. After
overnight liquid culture, cells are plated in methocult GF+ H4435,
Stem Cell Technologies) supplemented with cytokines (IL-3, IL-6,
IL-7, G-CSF, GM-CSF, CF, Flt3 ligand, and erythropoietin) and
various concentrations of known chemotherapeutic agents in
combination with either compounds of the present disclosure.
Colonies are counted by microscopy 12-14 days later. This method
can be used to test for evidence of additive or synergistic
activity.
Example 46: In Vivo Effect of Kinase Inhibitors on Leukemic
Cells
Female recipient mice are lethally irradiated from a .gamma. source
in two doses about 4 hr apart, with approximately 5 Gy each. About
1 hr after the second radiation dose, mice are injected i.v. with
about 1.times.10.sup.6 leukemic cells (e.g. Ph+ human or murine
cells, or p190 transduced bone marrow cells). These cells are
administered together with a radioprotective dose of about
5.times.10.sup.6 normal bone marrow cells from 3-5 week old donor
mice. Recipients are given antibiotics in the water and monitored
daily. Mice who become sick after about 14 days are euthanized and
lymphoid organs are harvested for analysis. Kinase inhibitor
treatment begins about 10 days after leukemic cell injection and
continues daily until the mice become sick or a maximum of
approximately 35 days post-transplant. Inhibitors are given by oral
lavage.
Peripheral blood cells are collected approximately on day 10
(pre-treatment) and upon euthanization (post treatment), contacted
with labeled anti-hCD4 antibodies and counted by flow cytometry.
This method can be used to demonstrate that the synergistic effect
of one or more compounds disclosed herein in combination with known
chemotherapeutic agents significantly reduce leukemic blood cell
counts as compared to treatment with known chemotherapeutic agents
(e.g. Gleevec) alone under the conditions tested.
Example 47: Treatment of Lupus Disease Model Mice
Mice lacking the inhibitory receptor Fc.gamma.RIIb that opposes
PI3K signaling in B cells develop lupus with high penetrance.
Fc.gamma.RIIb knockout mice (R2KO, Jackson Labs) are considered a
valid model of the human disease as some lupus patients show
decreased expression or function of Fc.gamma.RIIb (S. Bolland J. V.
Ravtech 2000. Immunity 12:277-285).
The R2KO mice develop lupus-like disease with anti-nuclear
antibodies, glomerulonephritis and proteinurea within about 4-6
months of age. For these experiments, the rapamycin analogue RAD001
(available from LC Laboratories) is used as a benchmark compound,
and administered orally. This compound has been shown to ameliorate
lupus symptoms in the B6.Sle1z.Sle3z model (T. Wu et al. J. Clin
Invest. 117:2186-2196).
Lupus disease model mice such as R2KO, BXSB or MLR/lpr are treated
at about 2 months old, approximately for about two months. Mice are
given doses of: vehicle, RAD001 at about 10 mg/kg, or compounds
disclosed herein at approximately 1 mg/kg to about 500 mg/kg. Blood
and urine samples are obtained at approximately throughout the
testing period, and tested for antinuclear antibodies (in dilutions
of serum) or protein concentration (in urine). Serum is also tested
for anti-ssDNA and anti-dsDNA antibodies by ELISA. Animals are
euthanized at day 60 and tissues harvested for measuring spleen
weight and kidney disease. Glomerulonephritis assessed in kidney
sections stained with H&E. Other animals are studied for about
two months after cessation of treatment, using the same
endpoints.
This model established in the art can be employed to demonstrate
that the kinase inhibitors disclosed herein can suppress or delay
the onset of lupus symptoms in lupus disease model mice.
Example 48: Murine Bone Marrow Transplant Assay
Female recipient mice are lethally irradiated from a .gamma. ray
source. About 1 hr after the radiation dose, mice are injected with
about 1.times.106 leukemic cells from early passage p190 transduced
cultures (e.g. as described in Cancer Genet Cytogenet. 2005 August;
161(1):51-6). These cells are administered together with a
radioprotective dose of approximately 5.times.106 normal bone
marrow cells from 3-5 wk old donor mice. Recipients are given
antibiotics in the water and monitored daily. Mice who become sick
after about 14 days are euthanized and lymphoid organs harvested
for flow cytometry and/or magnetic enrichment. Treatment begins on
approximately day 10 and continues daily until mice become sick, or
after a maximum of about 35 days post-transplant. Drugs are given
by oral gavage (p.o.). In a pilot experiment a dose of
chemotherapeutic that is not curative but delays leukemia onset by
about one week or less is identified; controls are vehicle-treated
or treated with chemotherapeutic agent, previously shown to delay
but not cure leukemogenesis in this model (e.g. imatinib at about
70 mg/kg twice daily). For the first phase p190 cells that express
eGFP are used, and postmortem analysis limited to enumeration of
the percentage of leukemic cells in bone marrow, spleen and lymph
node (LN) by flow cytometry. In the second phase, p190 cells that
express a tailless form of human CD4 are used and the postmortem
analysis includes magnetic sorting of hCD4+ cells from spleen
followed by immunoblot analysis of key signaling endpoints: p
Akt-T308 and S473; pS6 and p4EBP-1. As controls for immunoblot
detection, sorted cells are incubated in the presence or absence of
kinase inhibitors of the present disclosure inhibitors before
lysis. Optionally, "phosflow" is used to detect p Akt-S473 and
pS6-S235/236 in hCD4-gated cells without prior sorting. These
signaling studies are particularly useful if, for example,
drug-treated mice have not developed clinical leukemia at the 35
day time point. Kaplan-Meier plots of survival are generated and
statistical analysis done according to methods known in the art.
Results from p190 cells are analyzed separated as well as
cumulatively.
Samples of peripheral blood (100-200 .mu.l) are obtained weekly
from all mice, starting on day 10 immediately prior to commencing
treatment. Plasma is used for measuring drug concentrations, and
cells are analyzed for leukemia markers (eGFP or hCD4) and
signaling biomarkers as described herein.
This general assay known in the art may be used to demonstrate that
effective therapeutic doses of the compounds disclosed herein can
be used for inhibiting the proliferation of leukemic cells.
Example 49: TNP-Ficoll T-Cell Independent B-Cell Activation
Assay
To test the effects of the compounds of the present invention in
suppressing T cell independent antibody production, the TNP-Ficoll
B-cell activation assay was used as described herein. Compounds of
the present invention were dissolved in an appropriate vehicle
(e.g. 5% 1-methyl-2-pyrrolidinone, 85% polyethylene glycol 400, 10%
Solutor). Compounds were administered orally approximately 1 hr
before TNP-Ficoll treatment to 4-10 week old mice. To study the
effects of the compounds on B-cell activation, one set of mice were
grouped according to the following table:
TABLE-US-00005 Antigen Compound injection at Administration from
Mice/ Comp day-1 day-1 to day-7 Group# group treated Group TNP-F
Route (mg/kg) Route Regimen 1 4 Vehicle Antigen only 200 uL ip 0 Po
BID for 7 2 8 -- Antigen only (0.5 mg/ml) 0 days 3 8 Compound
reference 30 #7 4 8 Compound Antigen + cmp 1 5 8 #53 3 6 8 10 7 8
30 8 8 60
Four animals in group 1, and eight animals in groups 2 to 7 were
euthanized in CO.sub.2 2 hours after the last compound
administration on day 7. Blood was immediately collected by
cadio-puncture and kept at 37.degree. C. for 1 hr to clot followed
by overnight incubation at 4.degree. C. to allow the clot to
contract. The following day, serum was collected by decanting and
centrifugation at 3000 rpm for 10 min. The collected serum was then
frozen at -80.degree. C. for future analysis.
Serum samples were analyzed for anti-TNP antibody titers by ELISA
as described herein. TNP-BSA was coated onto a Nunc Maxisorb
microtiter plate with 100 .mu.l/well at a concentration of 10
.mu.g/ml in phosphate buffered saline (PBS). The Maxisorb plate was
incubated for 1.5 hours at room temperature and the solution was
removed. 200 .mu.l/well of blocking buffer (e.g. 1% BSA in PBS) was
added to each well and incubated 1 hr at room temperature. The
plate was washed once with 200 .mu.l/well of PBS 0.05% Tween-20
(wash buffer). A 1:2 dilution of serum from each mouse in blocking
buffer was added to each well in the first column (1) of the
microtiter plate. The serum in each well of column 1 was then
diluted 3-fold in blocking buffer and added to column 2. The serum
in each well of column 2 was diluted 3-fold in blocking buffer and
added to column 3. The procedure was repeated across the twelve
columns of the microtiter plate. The microtiter plate was incubated
1 hr at room temperature. Serum was removed from the plate and the
plate was washed three times with wash buffer. 100 .mu.l/well of
goat anti-mouse IgG3-HRP diluted 1:250 in blocking buffer was added
to each well and incubated 1 hr at room temperature. The anti-mouse
IgG3-HRP was removed from the microtiter plate and the plate was
washed six times with wash buffer. HRP substrate (200 .mu.l ABTS
solution+30% H.sub.2O.sub.2+10 ml citrate buffer) was added to each
well at 100 .mu.l/well, incubated 2-20 minutes in the dark and the
amount of anti-TNP IgG3 was determined spectrophotometrically at
405 nm. Similarly, anti-TNP IgM and total anti-TNP Ab were
determined using anti-mouse IgM-HRP and anti-mouse Ig-HRP
respectively.
The results as shown in FIG. 2 further show that under the
conditions tested compounds #7 and #53 exhibit 3.4 and 6.5-fold
reductions respectively in IgG3 levels relative to vehicle control
mice at a 30 mg/kg dose level. FIG. 2 further shows that compound
#53 exhibits 29.9-fold reduction in IgG3 levels relative to vehicle
control mice at a 60 mg/kg dose level under the conditions
tested.
Example 50: Rat Developing Type II Collagen Induced Arthritis
Assay
In order to study the effects of the compounds of the present
invention on the autoimmune disease arthritis, a collagen induced
developing arthritis model was used. Female Lewis rats were given
collagen injections at day 0. Bovine type II collagen was prepared
as a 4 mg/ml solution in 0.01N acetic acid. Equal volumes of
collagen and Freund's incomplete adjuvant were emulsified by hand
mixing until a bead of the emulsified material held its form in
water. Each rodent received a 300 .mu.l injection of the mixture at
each injection time spread over three subcutaneous sites on the
back.
Oral compound administration began on day 0 and continued through
day 16 with vehicle (5% NMP, 85% PEG 400, 10% Solutol) or compounds
of the present invention in vehicle or control (e.g. methotrexate)
at 12 hour intervals daily. Rats were weighed on days 0, 3, 6, 9-17
and caliper measurements of ankles taken on days 9-17. Final body
weights were taken, and then the animals were euthanized on day 17.
After euthanization, blood was drawn and hind paws and knees were
removed. Blood was further processed for pharmacokinetics
experiments as well as an anti-type II collagen antibody ELISA
assay. Hind paws were weighed and then with the knees preserved in
10% formalin. The paws and knees were subsequently processed for
microcopy. Livers, spleen and thymus were also weighed. Sciatic
nerves were prepared for histopathology.
Knee and ankle joints were fixed for 1-2 days and decalcified for
4-5 days Ankle joints were cut in half longitudinally, knees were
cut in half along the frontal plane. Joints were then processed,
embedded, sectioned and stained with toluidine blue. Scoring of the
joints was done according to the following criteria:
Knee and Ankle Inflammation
0=Normal
1=Minimal infiltration of inflammatory cells in
synovium/periarticular tissue
2=Mild infiltration
3=Moderate infiltration with moderate edema
4=Marked infiltration with marked edema
5=Severe infiltration with severe edema
Ankle Pannus
0=Normal
1=Minimal infiltration of pannus in cartilage and subchondral
bone
2=Mild infiltration (<1/4 of tibia or tarsals at marginal
zones)
3=Moderate infiltration (1/4 to 1/3 of tibia or small tarsals
affected at marginal zones)
4=Marked infiltration (1/2-3/4 of tibia or tarsals affected at
marginal zones)
5=Severe infiltration (>3/4 of tibia or tarsals affected at
marginal zones, severe distortion of overall architecture)
Knee Pannus
0=Normal
1=Minimal infiltration of pannus in cartilage and subchondral
bone
2=Mild infiltration (extends over up tot/4 of surface or
subchondral area of tibia or femur)
3=Moderate infiltration (extends over >1/4 but <1/2 of
surface or subchondral area of tibia or femur)
4=Marked infiltration (extends over 1/2 to 3/4 of tibial or femoral
surface)
5=Severe infiltration (covers >3/4 of surface)
Cartilage Damage (Ankle, Emphasis on Small Tarsals)
0=Normal
1=Minimal=minimal to mild loss of toluidine blue staining with no
obvious chondrocyte loss or collagen disruption
2=Mild=mild loss of toluidine blue staining with focal mild
(superficial) chondrocyte loss and/or collagen disruption
3=Moderate=moderate loss of toluidine blue staining with multifocal
moderate (depth to middle zone) chondrocyte loss and/or collagen
disruption, smaller tarsals affected to 1/2-3/4 depth
4=Marked=marked loss of toluidine blue staining with multifocal
marked (depth to deep zone) chondrocyte loss and/or collagen
disruption, 1 or more small tarsals have full thickness loss of
cartilage
5=Severe=severe diffuse loss of toluidine blue staining with
multifocal severe (depth to tide mark) chondrocyte loss and/or
collagen disruption
Cartilage Damage (Knee, Emphasis on Femoral Condyles)
0=Normal
1=Minimal=minimal to mild loss of toluidine blue staining with no
obvious chondrocyte loss or collagen disruption
2=Mild=mild loss of toluidine blue staining with focal mild
(superficial) chondrocyte loss and/or collagen disruption
3=Moderate=moderate loss of toluidine blue staining with multifocal
to diffuse moderate (depth to middle zone) chondrocyte loss and/or
collagen disruption
4=Marked=marked loss of toluidine blue staining with multifocal to
diffuse marked (depth to deep zone) chondrocyte loss and/or
collagen disruption or single femoral surface with total or near
total loss
5=Severe=severe diffuse loss of toluidine blue staining with
multifocal severe (depth to tide mark) chondrocyte loss and/or
collagen disruption on both femurs and/or tibias
Bone Resorption (Ankle)
0=Normal
1=Minimal=small areas of resorption, not readily apparent on low
magnification, rare osteoclasts
2=Mild=more numerous areas of resorption, not readily apparent on
low magnification, osteoclasts more numerous, <1/4 of tibia or
tarsals at marginal zones resorbed
3=Moderate=obvious resorption of medullary trabecular and cortical
bone without full thickness defects in cortex, loss of some
medullary trabecular, lesion apparent on low magnification,
osteoclasts more numerous, 1/4 to 1/3 of tibia or tarsals affected
at marginal zones 4=Marked=Full thickness defects in cortical bone,
often with distortion of profile of remaining cortical surface,
marked loss of medullary bone, numerous osteoclasts, 1/2-3/4 of
tibia or tarsals affected at marginal zones 5=Severe=Full thickness
defects in cortical bone, often with distortion of profile of
remaining cortical surface, marked loss of medullary bone, numerous
osteoclasts, >3/4 of tibia or tarsals affected at marginal
zones, severe distortion of overall architecture Bone Resorption
(Knee) 0=Normal 1=Minimal=small areas of resorption, not readily
apparent on low magnification, rare osteoclasts 2=Mild=more
numerous areas of resorption, definite loss of subchondral bone
involving 1/4 of tibial or femoral surface (medial or lateral)
3=Moderate=obvious resorption of subchondral bone involving >1/4
but <1/2 of tibial or femoral surface (medial or lateral)
4=Marked=obvious resorption of subchondral bone involving
.gtoreq.1/2 but <3/4 of tibial or femoral surface (medial or
lateral) 5=Severe=distortion of entire joint due to destruction
involving >3/4 of tibial or femoral surface (medial or
lateral)
Statistical analysis of body/paw weights, paw AUC parameters and
histopathologic parameters were evaluated using a Student's t-test
or other appropriate (ANOVA with post-test) with significance set
at the 5% significance level. Percent inhibition of paw weight and
AUC was calculated using the following formula: %
Inhibition=A-B/A.times.100 A=Mean Disease Control--Mean Normal
B=Mean Treated--Mean Normal
The results as shown in FIG. 3 demonstrate the effect of compound
#53 at 10, 30, and 60 mg/kg dosages at 12 hour intervals on mean
ankle diameter over time in a rat developing type II collagen
induced arthritis model under the conditions tested. Relative to
the vehicle alone control or to the methotrexate control, the
compounds of the present invention exhibited a significant
reduction in arthritis induced ankle diameter increase over
time.
The results as shown in FIG. 4 demonstrate the effect of compounds
#7 and #53 on ankle histopathology in the categories of
inflammation, pannus, cartilage damage, and bone resorption as
previously described under the conditions tested. The results show
a significant reduction in one or more categories by one of the
compounds of the present invention (i.e. compound #53) under the
conditions tested. FIG. 4 further shows that at 60 mg/kg, there is
a statistically significant reduction in all categories of ankle
histopathology for one of the compounds of the present invention
(i.e. compound #53) under the conditions tested. This suggests that
one or more compounds of the present invention may be useful for
the treatment and reduction of arthritis disease symptoms.
The results as shown in FIG. 5 demonstrate the effect of compounds
#7 and #53 on knee histopathology under the conditions tested. The
results demonstrate a dose dependent reduction in knee
histopathology. This suggests that one or more compounds of the
present invention may be useful for the treatment and reduction of
arthritis disease symptoms.
The results as shown in FIG. 6 demonstrate the effect of the
compounds #7 and #53 on serum anti-type II collagen levels under
the conditions tested. The results further show a significant
reduction at 10, 20, and 60 mg/kg dosage levels of serum anti-type
II collagen levels for compound #53, suggesting that one or more
compounds of the present invention may not only be useful for the
treatment and reduction of arthritis disease symptoms, but may also
be useful for the inhibition of the autoimmune reaction itself.
The results as shown in FIG. 7 demonstrate the effect of compound
#7 at 10, 30, and 60 mg/kg dosages at 12 hour intervals on mean
ankle diameter over time under the conditions tested. Relative to
the vehicle alone control or to the methotrexate control, the
compound exhibited a reduction in arthritis induced ankle diameter
increase over time under the conditions tested. When tested in the
same model, at least five other compounds of the present invention
exhibit comparable or even higher efficacy.
Example 51: Rat Established Type II Collagen Induced Arthritis
Assay
In order to examine the dose responsive efficacy of the compounds
of the present invention in inhibiting the inflammation, cartilage
destruction and bone resorption of 10 day established type II
collagen induced arthritis in rats, compounds were administered
orally daily or twice daily for 6 days.
Female Lewis rats were anesthetized and given collagen injections
prepared and administered as described previously on day 0. On day
6, animals were anesthetized and given a second collagen injection.
Caliper measurements of normal (pre-disease) right and left ankle
joints were performed on day 9. On days 10-11, arthritis typically
occurred and rats were randomized into treatment groups.
Randomization was performed after ankle joint swelling was
obviously established and there was good evidence of bilateral
disease.
After an animal was selected for enrollment in the study, treatment
was initiated by the oral route Animals were given vehicle, control
(Enbrel) or compound doses, twice daily or once daily (BID or QD
respectively). Dosing was administered on days 1-6 using a volume
of 2.5 ml/kg (BID) or 5 ml/kg (QD) for oral solutions. Rats were
weighed on days 1-7 following establishment of arthritis and
caliper measurements of ankles taken every day. Final body weights
were taken on day 7 and animals were euthanized.
The results as shown in FIG. 8 shows a significant reduction in
mean ankle diameter increase over time for compound #53 with a once
daily dosage under the conditions tested. The results in FIG. 9
futher demonstrate a significant reduction in mean ankle diameter
increase over time for compound #53 with a twice daily dosage under
the conditions tested. This suggests that the compounds of the
present invention can be useful for the treatment of autoimmune
diseases such as arthritis. When tested in the same model, at least
five other compounds of the present invention exhibit comparable or
even higher efficacy as compared to compound #53.
Example 52: Adjuvant Induced Arthritis Assay
Intrathecal Catheterization of Rats
Isoflurane-anesthetized Lewis rats (200-250 g) were implanted with
an intrathecal (IT) catheter. After a 6 d recovery period, all
animals except those that appeared to have sensory or motor
abnormalities (fewer than 5% of the total number) were used for
experiments. For IT administration, 10 .mu.l of drug or saline
followed by 10 .mu.l of isotonic saline was injected through the
catheter.
Adjuvant Arthritis and Drug Treatment
Lewis rats were immunized at the base of the tail with 0.1 ml of
complete Freund's adjuvant (CFA) on day 0 several days after
catheter implantation (n=6/group). Drug (e.g. one or more compounds
of the present invention or vehicle) treatment was generally
started on day 8 and continued daily until day 20. Clinical signs
of arthritis generally begin on day 10, and paw swelling was
determined every second day by water displacement
plethysmometry.
The results as depicted in FIG. 10 by the average change in paw
volume under the dosage regimes indicated show that under the
conditions tested, compound #53 shows a dose dependent reduction in
the average paw volume increase as measured in this adjuvant
induced arthritis model system. These results suggest that one or
more of the compounds of the present invention may be useful for
the treatment of one or more of the diseases or conditions
described herein.
The results as depicted in FIG. 11 show that compound #53 does not
exhibit toxicity or other adverse reaction under the conditions
tested as measured by a lack of weight loss.
Example 53: Rodent Pharmacokinetic Assay
In order to study the pharmacokinetics of the compounds of the
present invention a set of 4-10 week old mice are grouped according
to the following table:
TABLE-US-00006 Compound Administration Mice/ from day-1 to day-7
Group# group (mg/kg) Route Regimen 1 3 1 Po BID for 7 2 3 3 days 3
3 10 4 3 30 5 3 60
Compounds of the present invention are dissolved in an appropriate
vehicle (e.g. 5% 1-methyl-2-pyrrolidinone, 85% polyethylene glycol
400, 10% Solutor) and administered orally at 12 hour intervals
daily. All animals are euthanized in CO.sub.2 2 hours after the
final compound is administered. Blood is collected immediately and
kept on ice for plasma isolation. Plasma is isolated by
centrifuging at 5000 rpm for 10 minutes. Harvested plasma is frozen
for pharmacokinetic detection.
The results are expected to demonstrate the pharmacokinetic
parameters such as absorption, distribution, metabolism, excretion,
and toxicity for the compounds of the present invention.
Example 54: Basotest Assay
The baseotest assay is performed using Orpegen Pharma Basotest
reagent kit. Heparinized whole blood is pre-incubated with test
compound or solvent at 37 C for 20 min. Blood is then incubated
with assay kit stimulation buffer (to prime cells for response)
followed by allergen (dust mite extract or grass extract) for 20
min. The degranulation process is stopped by incubating the blood
samples on ice. The cells are then labeled with anti-IgE-PE to
detect basophilic granulocytes, and anti-gp53-FITC to detect gp53
(a glycoprotein expressed on activated basophils). After staining
red blood cells are lysed by addition of Lysing Solution. Cells are
washed, and analyzed by flow cytometry. Compounds 7 and 53 when
tested in this assay inhibit allergen induced activation of
basophilic granulocytes at sub micromolar range.
Example 55: Combination Use of PI3K.delta. Inhibitors and Agents
that Inhibit IgE Production or Activity
The compounds of the present invention may present synergistic or
additive efficacy when administered in combination with agents that
inhibit IgE production or activity. Agents that inhibit IgE
production include, for example, one or more of TEI-9874,
2-(4-(6-cyclohexyloxy-2-naphtyloxy)phenylacetamide)benzoic acid,
rapamycin, rapamycin analogs (i.e. rapalogs), TORC1 inhibitors,
TORC2 inhibitors, and any other compounds that inhibit mTORC1 and
mTORC2. Agents that inhibit IgE activity include, for example,
anti-IgE antibodies such as Omalizumab and TNX-901.
One or more of the subject compounds capable of inhibiting
PI3K.delta. are efficacious in treatment of autoimmune and
inflammatory disorders (AIID) for example rheumatoid arthritis. If
any of the compounds causes an undesired level of IgE production,
one may choose to administer it in combination with an agent that
inhibits IgE production or IgE activity. Additionally, the
administration of PI3K.delta. or PI3K.delta./.gamma. inhibitors of
the present invention in combination with inhibitors of mTOR may
also exhibit synergy through enhanced inhibition of the PI3K
pathway. Various in vivo and in vitro models may be used to
establish the effect of such combination treatment on AIID
including but not limited to (a) in vitro B-cell antibody
production assay, (b) in vivo TNP assay, and (c) rodent collagen
induced arthritis model.
(a) B-Cell Assay
Mice are euthanized, and the spleens are removed and dispersed
through a nylon mesh to generate a single-cell suspension. The
splenocytes are washed (following removal of erythrocytes by
osmotic shock) and incubated with anti-CD43 and anti-Mac-1
antibody-conjugated microbeads (Miltenyi Biotec). The bead-bound
cells are separated from unbound cells using a magnetic cell
sorter. The magnetized column retains the unwanted cells and the
resting B cells are collected in the flow-through. Purified B-cells
are stimulated with lipopolysaccharide or an anti-CD40 antibody and
interleukin 4. Stimulated B-cells are treated with vehicle alone or
with PI3K.delta. inhibitors of the present invention such as
compound 53 with and without mTOR inhibitors such as rapamycin,
rapalogs, or mTORC1/C2 inhibitors. The results are expected to show
that in the presence of mTOR inhibitors (e.g., rapamycin) alone,
there is little to no substantial effect on IgG and IgE response.
However, in the presence of PI3K.delta. and mTOR inhibitors, the
B-cells are expected to exhibit a decreased IgG response as
compared to the B-cells treated with vehicle alone, and the B-cells
are expected to exhibit a decreased IgE response as compared to the
response from B-cells treated with PI3K.delta. inhibitors
alone.
(b) TNP Assay
Mice are immunized with TNP-Ficoll or TNP-KHL and treated with:
vehicle, a PI3K.delta. inhibitor, for example, compound 53 of the
present invention, an mTOR inhibitor, for example rapamycin, or a
PI3K.delta. inhibitor in combination with an mTOR inhibitor such as
rapamycin. Antigen-specific serum IgE is measured by ELISA using
TNP-BSA coated plates and isotype specific labeled antibodies. It
is expected that mice treated with an mTOR inhibitor alone exhibit
little or no substantial effect on antigen specific IgG3 response
and no statistically significant elevation in IgE response as
compared to the vehicle control. It is also expected that mice
treated with both PI3K.delta. inhibitor and mTOR inhibitor exhibit
a reduction in antigen specific IgG3 response as compared to the
mice treated with vehicle alone. Additionally, the mice treated
with both PI3K.delta. inhibitor and mTOR inhibitor exhibit a
decrease in IgE response as compared to the mice treated with
PI3K.delta. inhibitor alone.
(c) Rat Collagen Induced Arthritis Model
Female Lewis rats are anesthetized and given collagen injections
prepared and administered as described previously on day 0. On day
6, animals are anesthetized and given a second collagen injection.
Caliper measurements of normal (pre-disease) right and left ankle
joints are performed on day 9. On days 10-11, arthritis typically
occurs and rats are randomized into treatment groups. Randomization
is performed after ankle joint swelling is obviously established
and there is good evidence of bilateral disease.
After an animal is selected for enrollment in the study, treatment
is initiated. Animals are given vehicle, PI3K.delta. inhibitor, or
PI3K.delta. inhibitor in combination with rapamycin. Dosing is
administered on days 1-6. Rats are weighed on days 1-7 following
establishment of arthritis and caliper measurements of ankles taken
every day. Final body weights are taken on day 7 and animals are
euthanized.
It is expected that the combination treatment using PI3K.delta.
inhibitor and rapamycin provides greater efficacy than treatment
with PI3K.delta. inhibitor alone.
While preferred embodiments of the present invention have been
shown and described herein, it will be obvious to those skilled in
the art that such embodiments are provided by way of example only.
Numerous variations, changes, and substitutions will now occur to
those skilled in the art without departing from the invention. It
should be understood that various alternatives to the embodiments
of the invention described herein may be employed in practicing the
invention. It is intended that the following claims define the
scope of the invention and that methods and structures within the
scope of these claims and their equivalents be covered thereby.
SEQUENCE LISTINGS
1
2112PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Glu Ala Ile Tyr Ala Ala Pro Phe Ala Lys Lys Lys
1 5 10 29PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 2Glu Ile Tyr Gly Glu Phe Lys Lys Lys 1 5
* * * * *
References